Other study sites included areas where no RVF activity was reported during the 2010–11 RVF outbreak. A total of 129 plots (30 m2) were selected where a visible difference could be seen in the wetland and upland vegetation. The Braun-Blanquet method was used for plant sampling. Classification was done using modified Two-Way Indicator Species Analysis. The vegetation analysis resulted in the identification of eight plant communities, seven sub-communities and two variants. Indirect ordination was carried out using CANOCO to investigate the relationship between species and wetland ecology. The study also identified 5 categories of wetlands including anthropogenic wetlands. Locations of reported RVF cases overlapped sites characterized by high clay-content soils and specific wetland vegetation. These findings indicate ecological and environmental parameters that represent preferred breeding habitat for RVFV competent mosquito vectors.Rift Valley fever (RVF) is endemic in Africa and parts of the Middle East. It is an emerging zoonotic disease threat to veterinary and public health. Outbreaks of the disease have severe socio-economic impacts. RVF virus emergence is closely associated with specific endorheic wetlands that are utilized by the virus’ mosquito vectors. Limited botanical vegetation surveys had been published with regard to RVF virus (RVFV) ecology. We report on a phytosociological classification, analysis and description of wetland vegetation and related abiotic parameters to elucidate factors possibly associated with the 2010–2011 RVFV disease outbreak in South Africa. The study sites were located in the western Free State and adjacent Northern Cape covering an area of ~40,000 km Rift Valley fever (RVF) is an emerging, arthropod-borne zoonotic disease  that couAedes spp. transmit the virus transovarially and the desiccation resistant eggs can survive long enough to maintain the virus between outbreaks [The ecology of RVF virus (RVFV) during inter-epidemic periods is poorly understood. Mostly based on a study by Linthicum et al. , it is gutbreaks . It appeutbreaks , 13, 14,utbreaks , sheep autbreaks , and in utbreaks , 18, basAedes and Culex mosquitoes.The association between abnormally high rainfall and RVF outbreaks has been documented by Anyamba et al.  and Sindet al. [Aedes species and sites of high RVFV activity.Prior to this study, no phytosociological investigations had been undertaken to explore the relationship between landscape-level ecology and reported RVF cases in livestock. This study compares wetland vegetation [et al.  and, moret al.  which liet al.  investigThe aims of this study were to: 1) conduct wetland vegetation surveys at sites of high RVF case mortality rates in livestock during the 2010 disease outbreak in the Republic of South Africa; 2) collect, describe and analyse biotic and abiotic environmental samples; and 3) combine the vegetation and environmental data to better understand the landscape-level ecology of RVFV.2  [The interior of South Africa in which the 2010 outbreak occurred, is arid to semi-arid with precipitation ranging from 450 mm in Bloemfontein, to less than 220 mm in the far west , 24. SpaOAA/CPC) .As shown in The study sites occur in the Grassland Biome  which coSelection and sampling of vegetation was conducted where a visible difference was seen in wetland vegetation found on shallow, isolated, non-saline depressions, littoral zones of open pans, and anthropogenic or riparian areas. Sample plots were located on a random basis within these units to ensure all vegetation variations were accounted for . The preAll plants were identified, vouchered, pressed and labelled according to standard, botanical field-techniques , a full Environmental samples included soil and water. Water temperature was taken using a standard laboratory mercury thermometer (range 100–0°C) on the surface of the water and at a 15 cm depth. Ad hoc, in-sun, ground, spot-temperatures were taken on various substrates using an infrared, hand-held thermometer (Major® MT 691 InfraRed thermometer).2 or 10 x 3 m2 to total 30 m2 to comply with theoretical criteria [Plot sizes varied according to wetland type, location and physical access to sites, and were estimated at 6 x 5 mcriteria  and estacriteria ). In allcriteria , 42, 43.Habitat and floristic data was captured using VegCap , with thAn initial approximation at clustering was conducted using TWINSPAN  algorithm of Hill  using JuThe wetland-indicator status of plants (the degree to which plant species associate with wetlands) was used , 49, whiObligate Wetland (OBL): estimated probability >99% in wetlands.Facultative Wetland (FACW): estimated probability 67% - 99% in wetlands.Facultative (FAC): estimated probability 34% - 66% in wetlands.Facultative Upland (FACU): estimated probability 67% - 99% occur outside wetlands, occasionally found in wetlands (estimated probability 1% - 33%).Obligate Upland (UPL): estimated probability >99% occur outside wetlands.Within Juice the lower threshold values for the diagnostic, constant and dominant species when applying the 'Analysis of Columns of Synoptic Tables'  functionThe naming of plant communities was done according to the standard system in current use in South Africa and according to the guidelines suggested by Brown et al. . The synTo achieve a normal distribution, the species data were log-transformed during ordination . Groups The skewness and kurtosis calculations performed with PC-ORD v5.0 revealed the non-unimodal distribution of the species data  eigenvalue of one for the first axis , 52. TheThe analysis of the 129 relevés using TWINSPAN incorporated in Juice produced 11 clusters. A final manipulation of relevé columns and species rows was done in Juice to fine-tune the phytosociological table, which was exported into Excel and refined for presentation by moving rows containing species and adding alphabetic letters to denote species groups . The synCyperus marginatus but have low species numbers as do most of the relevés comprising community 1. The gradient also indicates over-all low species numbers in response to low-rainfall, i.e. arid conditions and with low clay-content soils. An interpretation of the ordination diagramme  with 5 OBL species. Relevés 59, 54, 57, 67, 76, 64 on, or close to the horizontal axis, constitute a gradient indicative of semi-permanent to permanent inundation by fresh water, on low clay-content soils of low salinity. The outlier, relevé 64 has the two, tall, OBL species Cyperus longus (2), along with Agrostis lachnantha (2) which comprised, respectively, the dominant and diagnostic species for this community. The tight clustering of relevés 68, 72, 125, 58 and 122 at the centre of the diagram represents the 3-D overlay of Communities 6, 3 and 7 respectively illustrating the gradient of wetness represented by the numbers of OBL and FACW species decreasing to both the right and left.The outlier, relevé 71 (Community 6) has low species numbers and only two FACW plants unlike the rest of Community 6, defined by the 12 members of species groups P and Q , of whicOf the plants collected, 34% where Monocotyledons and 66% where Dicotyledons. There are 79 species that occur in ≤ 4 relevés and which do not form appreciable clusters, and have been left out of the formal phytosociological classification and description. However, as some of these may be habitat specialists, or endemics, all species are included in the synoptic table, Agrostis lachnantha, Sporobolus albicans and the low, spreading Cynodon dactylon, are dominant and diagnostic species. Sedges, Cyperaceae (21 species), comprise the most significant and dominant vegetation type, with the medium-height Cyperus laevigatus, C. longus, C. marginatus, and the tall, sharp-tipped Scirpoides dioecious the most important. Juncaceae, with Juncus exertus and J. rigidus forms a dominant component of about a third of the vegetation , then rush’s (Juncaceae), and thirdly, grasses (Poaceae) . Non-graposition  shows a Marisela capensis is an OBL species which appears on open, bare depression wetlands at the start of the growing season, and increases in cover until it produces fruiting bodies at the end of the wet period. It is a habitat indicator species preferring sandy clay soils, occurring with the low, spreading forb, Alternanthera nodiflora in Community 2, 3, 6 and Sub-Community 1.3 and forms associations with other members of Species Group I  pans. More recent and detailed work of Collins [Classification of pans and the associated wetland vegetation in the western Free State has previously been undertaken, covering an area of 41,819 kms region . Four pa Collins  and Muci Collins  adds to  Collins , that arFive categories of wetlands were identified; 1. Endorheic salt pans, 2. Non-saline depressions, 3. Palustrine wetlands, 4. Riparian wetlands, 5. Anthropogenic Wetlands. They are illustrated in The five-wetland-categorization was derived from field observations, and classified according to principles based on geology, soil colour and texture, vegetation-type, and physiognomy and presence/absence of surface water. The compilation in Aedes over the last 20 years (Kemp pers. Comm.). The category 3 wetlands  indicate wetland community on the margins of endorheic salt pans , mat-forming grass, also found in the variant 1.5.1 , [Floodwater , Kemp), . However, Kemp), . There i, Kemp), , 61, 62.Spot temperatures taken during the survey ranged from 18°C in the rain, taken on grass tufts, at 10.30 am, to the highest, in-sun temperatures of 71°C, on dead vegetation-matrix, at 11.35 am. The average was 42°C, on substrates varying from bare, grey soil, live grass tufts, vegetation matrix and mud .Juncus effusus, the sedges Cyperus laevigatus, C. marginatus, Scirpoides dioecious , and the grasses, Agrostis lachnantha (OBL), Cynodon dactylon, C. transvaalensis, Sporobolus albicans , Phragmites australis (OBL), Paspalum distichum (FACW), a wide-spread alien wetland grass species, and Eragrostis bicolor in the more arid areas. Significant wetland forbs include Pseudognaphalium luteo-album, Ranunculus multifidus, Rumex lanceolatus , and Veronica anagallis-aquatica (OBL).The vegetation and ecological study found 9 wetland plant associations characterised by the presence of hydrophilic grasses, sedges, rushes and forbs. The most species-rich communities occurred on the dolerite soils found in Bloemfontein which also receives the highest rainfall of all study sites. The wetland vegetation found on saline-soils underlain by shales producing high-clay content soils constitutes the majority of study sites and is coincident with the centre of the RVF 2010–2011 outbreak. The vegetation is dominated by OBL or FACW plants including; Bidens bipinnata, B. pilosa, Cirsium vulgare, Cosmos formosa, Oenothera rosa, Panicum coloratum, Paspalum distichum, Schkuhria pinnata, Tageties minuta, Verbena bonariensis  and Veronica anagallis-aquatica.Surprisingly few alien, invasive species where recorded, and include, An overlay of the geological map showing the South African Karoo Supergroup with the outbreak/wetland sites shows a striking correlation between the shales of the Ecca and Beaufort series, and the clay-rich, water-retaining soils produced from shale, the wetland types and the subsequent wetland vegetation. All these factors come together in the west/central Free State which has a plethora of these endorheic pans and Palaeo-Kimberley river wetlands which may provide appropriate habitat for RVFV mosquito vectors . The higAedes life-cycle . Markedly lower rates or zero livestock deaths occurred where the geology is not Karoo Supergroup sediments. Graspan/Holpan sites are on Andesite lava which erodes to a high iron content, red soil with little clay. Aedes were found at one of the small wetlands but in very low numbers, which may or may not be indicative of the suitability of these soils but could also be as a result of the limited rainfall at the time of the study.The other major environmental factor necessary for a RVF outbreak is sufficient, heavy rainfall to provide for the completion of the aquatic stages of the Aedes spp. vectors is most likely the single, major cause driving the emergence of RVFV. The two principal vectors in our study area are Aedes mcintoshi and Culex theileriAedes mcintoshi acts as primary or maintenance vector, capable of maintaining RVFV transovarially through desiccation-resistant embryonated eggs, and Culex theleri acts as secondary, amplifying vector [Culex vectors disperse widely to utilize anthropogenic wetlands and to feed on vertebrate hosts, leading to greatly enlarged mosquito populations. As a result, these Culex species are capable of extensive dispersal of RVF virus and amplification of the RVF outbreak [Flooding of the breeding habitat of RVFV-competent g vector . Primaryoutbreak , 63. ThiThe wetland vegetation and associated environmental parameters show the following gradients:Eragrostis bicolor with a few sedges and limited Juncus spp. The region has low records of RFV mortality in livestock, though Aedes mosquitoes have been found at Buffalo pan.The Graspan/Holpan area to the west in the Northern Cape, is the driest; precipitation < 250mm. The geology is Andesite, producing low-clay soils with limited water retention properties. The vegetation is dominated by the low grass Aedes. During this study, floodwater Aedes were collected at Buffalo pan, and in previous years at Mooigekry and Weltevrede , [The main study area; Reddersburg in the south, Bultfontein in the north with Brandfort, Dealsville, Petrusburg, Koffiefontein and Oppermansgronde centrally has moderate to high precipitation from 450mm-350mm. The geology is Karoo-Supergroup sediments, Beaufort sandstones with the grey shale horizon present throughout most of the site, which produces high clay-content soils. It is this central region of the Free State which has most of the endorheic salt pans, upland depressions resulting from the Palaeo-Kimberley River, and palustrine wetlands. It is also the region of highest recorded mortality for the 2010 outbreak. The vegetation is a component of the country-wide, azonal freshwater wetlands and is dominated by OBL and FACW wetland species which include sedges, grasses and rushes. It provides the ideal habitat for , 2016), , 64.Continued monitoring of the vegetation should continue using the Braun-Blanquet method for 2 reasons:to document the change in cover and,Aedes eggs surviving in wetland soils and vegetation. Understanding the complex link between rainfall, geology, soil structure, wetland type and the associated vegetation may provide information for management of floodwater Aedes spp. Populations in order to mitigate against catastrophic RFV outbreaks.to document the succession of plant species resulting from the change in climate due to the Walker Circulation, which initiated the development of La Niña conditions and a positive Southern Oscillation Index in late 2016. A better understanding of salinity, pH, water temperature and dissolved oxygen content is needed. Future studies should concentrate on acquiring more data to establish daily and seasonal variations, describe associations and examine for periodicity and patterns possibly responsible for In South Africa, high Rift Valley Fever mortality in the 2010 outbreak was concentrated in the region where numerous upland depression wetlands occurred. The geology of Communities 2, 3, 4, 5, 7 and 8 are all part of Ecca and Beaufort sandstone and shale which weather to produce high clay-content soils. These wetlands are also aligned with the Palaeo-Kimberley River system.Juncus and grasses, all of which are Obligate or Facultative Wetland species. It is known that the primary RVF Aedes vectors  breed in these areas and that, when flooded, the wetland would support the amplifying vectors of the Culex genus. Wetland vegetation provides the appropriate and (likely) ideal habitat for the floodwater species to lay eggs, hatch, and rest under the cool conditions provided by the vegetation while waiting to feed on and spread the virus to livestock that use the wetlands as watering points and for forage.This paper identified important characteristics of wetlands from areas where known RVFV mortalities have occurred (and that presumably were initiated by the mosquito vectors that use that habitat). All sites with RVF mortality had vegetation consisting of sedges, The vegetation and associated factors  found in community 2 to 8 wetlands, are likely condition for the emergence and propagation of RFV vectors. Of the 129 sample sites, Community 1 consisted of 27 relevés and constituted 21% of the total vegetation sampled. No records of RVF mortality were recorded from any of these sites. The vegetation contains almost no Obligate or Facultative Wetland species and is defined rather by species adapted to arid conditions in low-lying areas in which water accumulates after rain. The hydrogeomorphic conditions are distinctly different from those of the remaining 102 sample sites which have the common graminoid wetland vegetation.Juncus rigidus, Scirpoides dioecus, Sporobolus albicans and Cyperus laevigatus with communities 2 (12 relevés) and 5 (8 relevés), along with Cyperus longus, which is also a dominant and diagnostic species for community 6 (17 relevés). Communities 2, 5, and 6 constitute an additional 28.7% of the vegetation. All these species are Obligate or Facultative Wetland species and the 37 relevés from communities 2, 5, and 6 combined with the 49 from communities 7 and 8 comprise 66.7% of the total vegetation. The remaining vegetation found in community 4 (11 relevés) have anaerobic, hydrogeomorphic conditions suitable for Obligate and Facultative Wetland species, despite not having extensive or dense wetland vegetation such as found in communities 2, 5, 6, 7 and 8. All of the vegetation from communities 2, 5, 6, 7, 8 and the remaining from community 3 (11 relevés), and community 4 (3 relevés), had records of RVF mortality and combined, constitute 77.6% of the vegetation sampled at all the sites. Of all 129 sites sampled , 77.6% of all the vegetation, have documented cases of mortality due to RFV.Of the remaining sites, the most dense wetland vegetation was found in communities 7 and 8 and comprise 49 relevés which constitute 38% of the total vegetation sampled. Communities 7 and 8 share the dominant and diagnostic species of The wetlands and pans selected at each farm/park represent, phytosociologically, the overall vegetation found on the whole farm. And it is these pans and wetlands which are known breeding sites suitable for Aedes as primary vectors and which have secondary breeding sites for Culex, the amplifying vector for RFV virus.Aedes mosquitoes by identifying the most suitable mosquito breeding sites which could then be targeted to implement appropriate vector-control strategies to mitigate against catastrophic RFV outbreaks.Understanding the complex link between wetland vegetation, rainfall, geology, soils, and palustrine or wetland depression, may provide information for management of floodwater S1 Appendix(XLSX)Click here for additional data file.S2 Appendix(XLSX)Click here for additional data file.S3 Appendix(XLSX)Click here for additional data file.S4 Appendix(XLSM)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.


Cryptococcus neoformans (Cryptococcus species (Cryptococcus species, leading to a diagnosis of disseminated cryptococcosis involving lung and skin. The patient was treated with liposomal amphotericin B at a total dose of 4.5 g and then maintained on fluconazole 400 mg/week indefinitely, with total regression and healing of lesions.A 51-old-year male carpenter, who was a native of rural midwestern Brazil, presented with a 2-month history of a papule on the right malar region that had progressed to an ulcerated nodule. He had been diagnosed with Hansen’s disease 15 years prior and was under immunosuppressive therapy with prolonged courses of oral prednisone for treatment of his type 2 leprosy reaction. He had a history of environmental exposure to dense forests and areas of deforestation. Physical examination showed a 6-cm ulcerated nodule with an overlying crust on the right malar region, resembling leishmaniasis . His blooformans . Histolo species . Culture4Cryptococcosis occurs more commonly in immunocompromised hosts, including those with acquired immunodeficiency syndrome, Hodgkin’s lymphoma, or those who use immunosuppressive therapies including corticosteroids.Cryptococcus neoformans is a yeast-like encapsulated fungus, which is typically found in pigeon droppings.2 The main portal of entry is the respiratory tract, and lungs are the primary site of infection. In most instances, the infection is subclinical and self-limiting.2 However, the infection may progress by hematogenous dissemination to any part of the body.2 The skin is the most common site of dissemination, and cutaneous lesions occur in 5% to 15% of patients.3 The lesions may occur as ulcers, pustules, granulomata, abscesses, or herpetiform or molluscum contagiosum-like lesions.5 These skin lesions may simulate a variety of diseases, such as cellulitis, histoplasmosis, or basal cell carcinoma.5 In the case reported here, the lesion was an ulcerated nodule, mimicking cutaneous leishmaniasis. In addition to biopsies, the diagnosis may be made by other direct means such as with Tzanck smears, India ink, or potassium hydroxide examinations.5 Cryptococcosis can clinically mimic leishmaniasis, and the possibility that an ulcerated nodule in immunocompromised hosts may be caused by C. neoformans should be considered.


To observe and analyze the application effect of ultrasound-guided modified Seldinger technique (MST) in Peripherally Inserted Central Catheter (PICC) catheterization.Two hundred patients treated with PICC catheterization from January 2013 to December 2015 were selected and randomly divided into two groups, namely, observation group and control group. The observation group adopted ultrasound-guided MST for catheterization while the control group applied traditional puncture technique for catheterization. Then efficacy of catheterization, success rate of catheterization and incidence rates of complications were compared between two groups.Various indicators of catheterization effects of the observation group were better than those of the control group, and the differences were statistically significant (P<0.05); one-time success rate of puncture and catheterization of the observation group was both higher than the control group (P<0.05);. Moreover, the incidence of puncture points bleeding, phlebitis and thrombus were all lower than those of the control group (P<0.05).Implementing PICC catheterization based on ultrasound-guided modified Seldinger puncture technique can increase success rate of puncture, improve the effect of catheterization, lower incidence rate of adverse effects of catheterization and improve satisfaction and comfort level of patients. Peripherally Inserted Central Catheter (PICC) makes quick dilution of intravenous dripping drugs possible and is capable of reducing stimulus of drugs to blood vessels. Also, it can prevent patients from suffering from pain induced by repeated puncture and intensely irritant drugs. For long-term venous transfusion, and process of intermittent treatment, PICC is recognized as a safe, economical and reliable venous transfusion way, which causes little trauma, and it is widely used in clinical practices.4In China, vessel puncture is a conventional puncture technique of PICC embedding, which requires thick, straight, and elastic blood vessels. For those patients who are in urgent need of catheterization but with poor vein conditions, traditional PICC puncture has disadvantages for it is time consuming, and might cause great damage to local tissues.Two hundred patients who were treated with PICC catheterization in Binzhou People’s Hospital from Jan. 2013 to Dec. 2015 were selected as research objects. Patients who had venous transfusion time≥6 d, have underwent chemotherapy, were conscious with good cognitive abilities, were aware of research contents and volunteered to sign informed consent form were included. They were divided into two groups, observation group (N=100) and control group (N=100), according to random number table. There were 60 males and 40 females in the observation group, aged from 20 to 69 years (average (52.1±3.4) years), among which there were 42 cases of lung cancer, 33 cases of breast cancer, 19 cases of stomach cancer, and 6 cases of liver cancer. In the control group, there were 63 males and 37 females, aged from 21 to 70 years (average (53.1±3.7) years of age). It included 40 cases of lung cancer, 32 cases of breast cancer, 23 cases of stomach cancer and 5 cases of liver cancer. Differences of general data between of two groups were not statistically significant (P>0.05); hence the results were comparable.Traditional puncture method (blind puncture) was adopted: operators evaluated blood vessels of lower arm and marked the puncture points through visual inspection, touch or according to clinical experiences of puncture. The operator used left hand to fix skin of puncture point, and used right hand to insert needle into blood vessels of puncture point at the angle of 30° by right hand using 16G steel puncture needles. When saw the blood returned, the operator pulled out the needle and put the catheter to expected location by puncture sheath.Ultrasound-guided MST puncture method was adopted: We selected GE vivid e9 ultrasound equipment and Seldinger puncture suite produced by General Electric Company. Patients were in horizontal position, and the catheterized limbs extended at the angle of 60° to 90°. Then we used vascular ultrasound instruments to select proper veins above elbow and mark them. And we measured the length of catheters that were to be inserted, routinely sterilized the catheterization parts, put sterile cover over probe and smeared sterilizing coupling agents. The assistant tied tourniquet, while the operator inserted puncture needle into blood vessels from marked puncture points. After blood returned, the operator used color doppler ultrasound to observe whether blood vessels were unobstructed and pushed into outer cannulas and polled out corn of needle. Next, the guiding wire was pushed for 5-10 cm with tourniquet tied. If the delivery was unhindered, the tourniquet was untied and the guiding wire was pushed for 15 cm. After outer cannulas were drawn out, the puncture point was covered with gauze. Lidocaine was injected subcutaneously around puncture points, and needle eye was pressed with gauze for 5-10 s. Then the truncated edge of skin expander was cling to tip of guiding wire and was put into hypoderm for 0.3-0.5 cm, to expand the incision. Next, the vascular dilator and leading-in sheath components were put to skin incision through the end tail of guiding wire, and the leading-in sheath was put into the incision. After conforming by ultrasound that the leading-in sheath had entered into a blood vessel, the dilator and leading-in sheath were separated, and the dilator and the guiding wire were taken out. After ultrasound suggested PICC catheters had entered into veins, the supporting guiding wire was taken out, and was routinely fixed. At last, the location of the tip of the catheter was identified using X-ray.(1) Effects of catheterization: this indicator mainly included achievement of expected goals, comfort levels, pain score, and rate of puncture failure; (2) Success rate of catheterization: this indicator mainly included success rate of puncture  and success rate of catheterization ; (3) Incidence rates of complications: this indicator mainly included incidences rates of puncture site bleeding, phlebitis, and thrombus 2 months after puncture.The first criterion was comfort level. Comfort level questionnaire developed by our hospital was used for investigation. The questionnaire was designed by selecting 10 issues which was concerned most by patients based on literature. The score of each item was 1 ~ 5 points. A total score of 45 ~ 50 points was determined as comfort, 25 ~ 34 points as general, 15 ~ 24 points as discomfort, and less than 15 points as quite uncomfort, i.e., higher score meant higher comfort level. The questionnaire was evaluted with a content validity of 0.81 and 0.856 Cronbach’α coeffcient by five clinical experts, suggesting its good reliability and validity. The second criterion was pain score. The degree of pain was scored using numerical rating scale. A straight line with a length of 10 cm was divided into 10 pieces evenly, marked as 0 ~ 10 points respectively. Patients marked the line by himseves according to the pain they suffer. Higher score meant more serious pain. 0 point stands for no pain, 1 ~ 3 points stands for mild pain, 4 ~ 6 points as medium pain, and higher than 7 points as severe pain.All data were analyzed by SPSS 20.0 statistical software, and the measurement data were expressed by mean±SD. The comparisons were tested by using t-test, while the enumeration data were expressed by percentage and the comparisons were tested by chi-square test. If P<0.05, the differences were considered as statistically significant.Various indicators of catheterization effects of two groups of the observation group were better than those of the control group. Among the observation group, there were 93 cases whose treatment effects met expectation while in the control group, there were only 64 cases. Failure rate of the observation group was only 2.0%, which was obviously lower than the control group (34%); Pain score of the observation group was lower than that of the control group, and the comfort levels score of the former was higher than the latter. The differences of the indicators between two groups were statistically significant (P<0.05). One-time success rate of puncture and one-time success rate of catheterization of the observation group were higher than those of the control group, and the differences were statistically significant (P<0.05). The incidence of puncture points bleeding, phlebitis and thrombus of the observation group was all lower than those of the control group (P<0.05). As to the observation group, the one-time, two-time, three or higher-time success rate of catheterization of patients with 2.0 ~ 2.5 mm vescular diameter was 20.0%, 60.0% and 20.0% respectively; the success rate of patients with 2.6 ~ 3.0 mm vascular diameter was 85.7%, 9.5% and 4.8% respectively; the success rate of patients with 3.1 ~ 4.0 mm vascular diameter was 91.7%, 8.3% and 0; the success rate of patients with 4.0 mm larger vascular diameter was 100%, 0% and 0%. It indicated that, patients with larger vascular diameter had higher one-time success rate of cauterization.Traditional puncture methods adopt means of touching and external visual inspection. Some of the methods evaluate the conditions of blood vessels according to experience before puncture. However, for patients who are fat, with flabby skins or with poor vessel conditions, it is difficult for traditional blind puncture to achieve one-time success.A recent study showed that, patients who received traditional puncture methods might be required to draw out catheters because of unplanned catheters drawing, high incidence rate of complications, etc, during later period of therapeutic process with catheters, which greatly influenced the comfort levels of treatment period with catheters.Moreover, results of the study showed that, incidence of puncture points bleeding and phlebitis of the observation group were lower than those of the control group. Traditional PICC catheterization puncture does great damage to vessels and leads to incidence of complications such as phlebitis and puncture points’ bleeding.To sum up, using MST to implement PICC catheterization under the guidance of high-definition and visual vascular ultrasound provides cancer patients with safe and effective intravenous indwelling channels. Due to the catheterization positions moving from below elbow to above elbows, applications of vascular ultrasound systems, and updates of puncture technique, occurrences of complications after PICC catheterization were greatly reduced, which ensured the success of catheterization. Compared to traditional blind puncture technique, MST is characterized by low incidence of complications during catheterization, high comfort levels, low failure rate of puncture, low pain scores, etc. Thus, it can be regarded as the preferred puncture scheme for patients who require long-term transfusions, for it is significantly effective and deserved to be clinically generalized and applied.


NekNomination, also known as NekNominate, Neck and Nominate, or Neck Nomination, is a social network–based drinking game which is thought to have originated in Australia and spread all over the world between 2013 and 2014. Individuals record videos of themselves while rapidly drinking excessive quantities of alcoholic drinks (necking) and then nominate friends to outdo them within 24 hours; the videos are then posted on social media such as Facebook or YouTube. The consequences of this drinking game have been very dangerous; at least 5 people under age 30 years have died after drinking deadly cocktails, and many others have suffered from alcohol intoxication.The goal of the research is to evaluate data about clinically important acute alcohol intoxication among teenagers and young adults and inform and educate the general public, especially parents, teachers, and health workers, about the spreading craze of dangerous Internet-related behavior among today’s teenagers and young people up to the age of 23 years.Patients aged 15 to 23 years with acute alcohol intoxication who came to the emergency department (ED) of 2 major hospitals in Italy from January 1, 2011, to June 30, 2014, were included in this study. Data were retrieved from prehospital and intrahospital medical records and included personal information, methods of intoxication, triage color code, date and time of access to the ED, any relevant signs and symptoms, blood alcohol concentration, and diagnosis at discharge.A total of 450 young patients  were recruited. The causes of intoxication were happy hour, binge drinking, NekNominate, eyeballing, other alcoholic games, or a mix of them. Happy hour was found to be more common among the older patients, whereas NekNominate accounted for almost half of the youngest group of hospitalizations. Eyeballing occurred in 1.6% (7/450) of cases; binge drinking and other alcoholic games caused 23.3% (105/450) and 23.8% (107/450) of hospitalizations, respectively. On admission, 44.2% (199/450) of patients were assigned a red or yellow color code requiring immediate medical attention; about 14% of them required additional medical assistance (after being in the ED) or hospitalization, some in semi-intensive care units.Our study shows that the increased numbers of hospitalizations due to alcohol intoxication in the adolescent age group, as a consequence of NekNominate or other drinking games, is alarming and represents a serious public health issue. The potential markers of improper use of social networks must be clearly identified, including categories at risk of alcohol abuse, in order to develop intervention and prevention strategies in terms of education and awareness, which may help in averting potentially fatal episodes. These episodes very often ended up in brawls [Drinking games are thought to have originated thousands of years ago, when the ancient Romans played “ n brawls .The common concept now is that drinking games are: (1) social drinking events, 2) played according to a set of rules specifying when and how much players should drink, (3) designed to promote the rapid consumption of large amounts of alcohol to facilitate drunkenness, and (4) involve the drinkers in performing cognitive and/or motor tasks [ played aPolizzotto et al  classifiDrinking games inevitably promote heavy alcohol consumption  and, comSince 1995, binge drinking has been increasing among teenagers aged 15 to 16 years in Europe . About 1Alternative drinking games practiced by young people have recently appeared. NekNominate is one of them, as well as “kings,” “flip cup,” “beer roulette,” “beer pool,” “tequila roulette,” “speed pennies,” “fuzzy duck,” and many others. “Butt chugging,” also known as “enema alcohol,” is the introduction of alcohol directly into the rectum and colon through the anus ; the rec“Eyeballing” is performed by pouring alcoholic substances directly into the eyes; substances with high alcohol content are generally preferred, although cases of misuse of other substances  have also been reported . DrinkinSome of these practices are known; others have not been documented in medical journals or have only been discussed as case reports giving basic information . The popPeer pressure is a well-known, influential fuel for excessive alcohol consumption, and Web-based social networks now offer a platform to expand the extent and impact of peer-to-peer coercion . Mu et aNekNominate is a novel game that involves individuals posting videos of rapid alcohol consumption (“necking”) on social media sites and nominating an acquaintance to exceed their behavior within 24 hours. The named individual will then drink a larger quantity or a more extreme mixture with associated illegal, risky, embarrassing, or self-injurious behaviors. Other substances are often added to the alcohol and have included eggs, goldfish, insects, rodents, battery fluid, motor oil, de-icer, and human urine; associated behaviors have included driving, biking, skateboarding, and swimming while drinking . What drMedia reports have identified 5 NekNominate-related fatalities in the United Kingdom and Republic of Ireland, all occurring in February 2014 among young men aged 19 to 29 years; 4 from presumed acute alcohol poisoning and 1 from drowning after drinking . HoweverThe objective of our research is to evaluate data about clinically relevant  acute alcohol intoxication episodes among teenagers and young adults, while focusing on the methods of reaching drunkenness  by analyzing ED admissions. In addition, we want to inform and educate the general public, especially parents, teachers, and health workers, about the rapid spread of dangerous Internet-related behaviors among today’s teenagers.This research is a retrospective longitudinal observational single-center study conducted at two major hospitals in Italy. Young patients who accessed the EDs of these hospitals from January 1, 2011, to June 30, 2014, were included in this study. Inclusion criteria are aged 15 to 23 years, diagnosis of acute intoxication by blood test, and positive history of alcohol intoxication due to happy hour, binge drinking, Neknominate, eyeballing, other alcoholic games, or a mix of them, with or without drunkenness-related injuries. Patients who are younger than 15 years or older than 23 years, have no diagnosis of acute alcohol intoxication, or have a negative history for above-mentioned alcohol-related activities are excluded from the study.Patients included in this study were asked (directly or through their relatives or friends at the ED) for personal information and methods of intoxication. Also recorded for the sample of selected patients were triage color-code , the date and time of access to the ED, any relevant signs and symptoms, blood alcohol concentration , and discharge diagnosis. Patients were classified by gender (M/F) and age group .Considering the inclusion and exclusion criteria, 450 young patients who accessed the ED for acute alcohol intoxication due to specific drinking behaviors were recruited. The reasons for their intoxication were happy hour, binge drinking, NekNominate, eyeballing, other alcoholic games, or a mix of these. Among these patients, 15 (3.3%) were aged 15 to 16 years, 184 (40.9%) were 17 to 18 years, and 251 (55.8%) were 19 to 23 years; in total, there were 277 boys (61.5%) and 173 (38.5%) girls. The different distribution of alcoholic consumption classified by drinking behavior and age group in the general population is listed in Generally, young adults accessed the ED during the weekend at night, and alcohol-related hospitalizations therefore had an important effect on ED personnel, particularly on weekends. Conversely, Neknomination cases in our sample of adolescents accessed the ED during afternoons between Monday and Friday and during evening hours from Wednesday to Sunday. Half  of the patients were admitted between 12 AM and 8 AM.None of the 15- to 16-year-old patients had alcohol intoxication while attending a happy hour event, but 30.4% (56/184) and 24.7% (62/251) of the 17- to 18- and 19- to 23-year-old groups, respectively, got drunk during happy hour. NekNominate-related hospitalizations totaled 5.8% (26/450) in the entire sample and represented almost half of the youngest group of hospitalizations. Eyeballing accounted for 1.6% (7/450) cases; binge drinking and other alcoholic games caused 23.3% (105/450) and 23.8% (107/450) of hospitalizations, respectively. A total of 53.2% (98/184) of the patients aged 19 to 23 years had been driving under the influence of alcohol during the late hours of the weekend.BAC in the population varied between 1.5 and 2.4 g/L; 56% (252/450) of patients stated that they had consumed an excessive amount of alcohol in a short period of time. The most common signs and symptoms included heart palpitations , headache , diarrhea , nausea and vomiting , anxiety , problematic behavior , hallucinations , convulsions , coma , unconscious state , slow respiration , and rectal edema and severe acute colitis .On hospital admission, 44.2% (199/450) of the patients were assigned a red or yellow color code, requiring immediate medical attention; 4.4% (20/450) were transferred to the short-term medical observation ward because they required continuous clinical evaluation, diagnostic testing, and appropriate treatment protocols; 3.3% (15/450) were admitted to a semi-intensive care unit; 80.4% (362/450) were discharged within 72 hours; 6.0% (27/450) were hospitalized; 2.0% (9/450) were spontaneously discharged; and 4.3% (18/450) refused hospitalization.To our knowledge, this is the first study analyzing the clinical consequences of drinking games, with particular attention to NekNominate as a social media–based phenomenon. Some of these practices are already known; others have not been documented in medical journals or have only been discussed as case reports giving basic information. Drinking behaviors including the alcoholic games Neknomination and eyeballing are still not well recognized as public health problems, but increasing numbers of adolescents are admitted to EDs for acute alcoholic intoxication.Our sample contained 450 young patients aged 15 to 23 years who accessed the ED after ingesting excessive amounts of alcohol in an unconventional and dangerous way. Most of them were between 19 and 23 years old and male. Of all patients, happy hour  was the most common way of reaching severe drunkenness , followed by various alcoholic games, binge drinking, mixed behaviors, NekNomination and eyeballing. Happy hour only affected patients aged 17 to 23 years, indicating that this practice is not frequent among younger people. Conversely, NekNomination was the most common cause of alcohol intoxication among the youngest patients (aged 15 to 16 years), representing 47% of all hospitalizations in this age group. This figure adds to the dangerousness of this practice in view of the fact that the earlier alcohol abuse begins, the worse the consequences . In addAlcoholic games in general have caused increasing numbers of hospitalizations directly proportional to patients’ ages: 13% in the 15- to 16-year-old group, 16% in those aged 17 to 18 years, and 30% in those aged 19 to 23 years, and this matches the most recent reviews about the correlation of age with drinking games . Most ofThe abuse of alcoholic drinks is a significant source of risk for adolescents and is one of the primary causes of many accidental injuries, such as falls, accidents at home, and road accidents . A large proportion (44%) of patients admitted to the ED were assigned a red or yellow color code, meaning that they required immediate or almost immediate treatment; about 14% of them required additional medical assistance (after being in the ED) or hospitalization, sometimes in a semi-intensive care unit, representing a significant burden in terms of personnel requirements and costs for diagnosis and treatments. The most common presenting signs and symptoms were those typical of alcohol intoxication: heart palpitations (66%), headache (55%), diarrhea (44%), nausea and vomiting (66%), and problematic behavior (63%).NekNominate seems to be an on-off phenomenon related to 2013-2014; nevertheless, Wombacher et al  wrote abWe present the various patterns of alcohol intoxication among adolescents with the image of an iceberg to highlight the magnitude of unknown behaviors compared with what is known in the scientific literature . There aMost of the iceberg is below the waterline and represents people’s unawareness regarding young people’s acute alcohol consumption. Binge drinking, Neknomination, and alcoholic games are also often associated with abnormal behaviors such as not wearing a helmet while riding a motorbike, not fastening a seatbelt while driving a car , having promiscuous sex, and consuming illicit drugs.The widespread use of social media is having a severe effect on our society: its easy accessibility makes it one of the most commonly used tools among teenagers and young adults, and the changes induced in current behaviors are still under evaluation . Social This study is limited by the small sample size, which is not representative of the entire Italian teenager population. However, it is still useful for preliminary data about drinking games.Drinking games are still relatively undisclosed and “at the bottom of the iceberg” of health workers’ attention and knowledge. They encourage young people to consume large quantities of alcohol within a short period of time, putting them at risk of alcohol intoxication that can potentially cause injuries, later regretted sexual activities, suicides, and traffic accidents and deaths.Social media is an important tool for connections among young people and may be associated with behavioral and social maladjustment among adolescents. It also represents a powerful source of peer pressure. Adolescents who consume alcohol in new and dangerous ways such as NekNomination reach EDs with high BAC, are classified as red or yellow codes at triage, present severe impairment of vital functions, and often require further intensive medical assistance.In order to develop a prevention strategy which could help to avert potentially fatal episodes, it is necessary to highlight the potential markers of problematic social network use and to inform and educate health personnel, social workers, and school teachers about these new phenomena, which are spreading very rapidly among adolescents. Further studies to better describe and analyze the problem, particularly in its clinical aspects, are in progress.


Intensity and duration of physical activity are associated with the achievement of health benefits. Our aim was to characterize physical activity behavior in terms of intensity, duration pattern, and adherence to the WHO physical activity recommendations in a population-based sample of adults from southern Germany. Further, we investigated associations between physical activity and sex, age, and body mass index (BMI), considering also common chronic diseases.We analyzed 475 subjects  who wore ActiGraph accelerometers for up to seven days. Measured accelerations per minute obtained from the vertical axis  and the vector magnitude of all three axes  were classified as sedentary, light or moderate-to-vigorous physical activity (MVPA) according to predefined acceleration count cut-offs. The average minutes/day spent in each activity level per subject served as outcome. Associations of sex, age, BMI, and seven chronic diseases or health limitations, with the activity levels were analyzed by negative binomial regression.Most of the wear time was spent in sedentarism (median 61%/day), whereas the median time spent in MVPA was only 3%, with men achieving more MVPA than women . Almost two thirds of MVPA was achieved in short bouts of less than 5 minutes, and 35% of the subjects did not achieve a single 10-minute bout. Hence, only 14% adhered to the WHO recommendation of 2.5 hours of MVPA/week in at least 10-minute bouts. Females, older subjects and obese subjects spent less time in MVPA (p<0.05), but no clear association with hypertension, asthma, diabetes, chronic obstructive pulmonary disease, anxiety/depression, pain or walking difficulties was observed in regression analyses with MVPA as outcome.Activity behavior among middle-aged German adults was highly insufficient, indicating a further need for physical activity promotion in order to gain health benefits. Being physically active is linked to several health benefits, including the improvement of functional ability, cardiorespiratory fitness, metabolic health, and the prevention of premature death –4. AlthoIn order to improve adherence and to establish PA promotion, it is important to collect evidence on the pattern of PA behavior, however assessing PA is challenging –7. VarioLarge scale, population-based data on PA in German adults was provided from the health monitoring system at the Robert Koch Institute based only on self-reports. Among 7671 participants, aged 18–79 years, 20% reported to achieve the WHO threshold of at least 2.5 hours of MVPA/week . FurtherThe aim of the present study was to determine the PA levels and PA duration pattern assessed by accelerometry in a German adult cohort aged 48–68 years. Duration patterns of interest were the time spent in MVPA assessed in various bouts and the adherence to the WHO PA recommendations for adults categorized by sex and age. A further goal was to analyze the associations of sex, age, and BMI with activity levels serving as outcome. Since evidence indicates that subjects with chronic health issues engage less in PA , 17, we The present analysis was based on a follow-up study of the KORA  S4 cohort comprising 4261 adults examined in 1999–2001. The primary study design has been described previously . BetweenBaseline information on sociodemographic variables, self-reported physician diagnosis of common chronic diseases such as asthma or diabetes, and medication use was obtained during a standardized interview, also including questionnaires such as the EuroQol—5 Dimensions (EQ-5D) questionnaire and the shortened medical outcome survey (SF-12) which are assessment tools for health status and health-related quality of life. Further, participants underwent a standardized medical examination. Height and weight were assessed while subjects were wearing light clothes without shoes. After a rest of at least 5 minutes in a sitting position, blood pressure was measured three times on the right arm and determined by the mean of the second and third measurement.In the present analysis, diabetes was defined based on self-reported physician diagnosis or use of antidiabetic agents. Hypertension was defined as blood pressure greater or equal to 140/90 mmHg or if the subject reported the use of antihypertensive medication given that the subject was aware of being hypertensive. Subjects who reported a doctor’s diagnosis of chronic bronchitis or chronic obstructive pulmonary disease (COPD) were classified as having COPD. Information on mobility, pain/physical complaints and anxiety/depression was derived from the EQ-5D, and on overall health from the SF-12 questionnaire.The subjects received the accelerometer, instructions on its use, and a diary sheet at the study center. Participants were asked to start wearing the monitor on the following morning immediately after getting out of bed, and to complete a daily diary  which inMeasured accelerations primarily sampled at 30 Hz and stored at 1 Hz were resampled in one minute epochs for further data analysis. As recommended by ActiGraph, data filtering was set to default . 1-minute epochs were chosen to allow for comparisons with other population-based studies , 12–14 aAccording to the recorded acceleration value per minute, each minute was classified into one of three activity levels  using two approaches: the uniaxial count cut-offs of the vertical axis were based on the commonly used Freedson et al.  cut-offsth and 75th percentiles.Pearson’s Chi-square test, Fishers exact test (if cell count <5), and Wilcoxon rank-sum test  were used to assess sex specific differences, group differences in the analyzed study population, and differences between subjects designated to participate in the “Lung health & physical activity” section and those who participated in the PA assessment. Time counts of all three activity levels were averaged over the reporting period for each subject and described by median and the 252) was calculated using values of height and weight obtained by physical examination in the study center. Subjects were categorized as normal weight (BMI 18–25 kg/m2), overweight (BMI ≥25 kg/m2), and obese (BMI ≥30 kg/m2) [Among the study population, age, ranging from 48 to 68 years, was grouped into three age tertiles . BMI (kg/m0 kg/m2) .Mean minutes of MVPA/subject showed a skewed distribution, with a great variance. Negative binomial regression, which accounts for overdispersion, was applied to investigate the associations between time spent in each PA level  with sex, age and BMI.Sedentary, light and MVPA served as outcome in all analyses. Each outcome was expressed in minutes averaged over the recording period for each subject and rounded to the nearest integer. For each outcome, a basic model was applied, which was adjusted for sex, age and BMI. Stepwise selection (combined forward and backward selection), using the lowest Akaike information criterion (AIC) as stopping rule, was performed to determine a model for each activity level considering several covariates. Besides sex, age and BMI, the following covariates were assessed: (1) season, categorized as winter (start of measurement: December to February), spring (March to May), summer (June to August), and autumn (September to November), (2) education, categorized as low (<10 years of school), middle (10 years of school) and high (>10 years of school), (3) hypertension, (4) diabetes, (5) asthma, (6) COPD, (7) difficulties with walking, (8) pain or physical complaints, and (9) anxiety or depression. All models with sedentary and light activity as outcome were additionally adjusted for average wear time/day. Our study population comprised Caucasians only, so ethnicity was not considered as a covariate.A potential interaction effect of sex was tested in the final models. Sensitivity analyses were performed through exclusion of subjects reporting fair/poor overall health (N = 49) and exclusion of subjects who reported myocardial infarction (N = 14) and/or stroke (N = 8), to avoid possible bias towards a lower PA due to these diseases. All analyses were performed with uniaxial cut-offs. To assess the impact of uni- vs. triaxial motion monitoring and to ensure better comparability to other studies, we replicated all aforementioned analyses using triaxial cut-offs.Adherence to the WHO PA recommendation was defined as achieving at least 2.5 hours of MVPA/week in bouts of at least 10 minutes. Therefore, hours of MVPA/day spent in bouts of at least 10 minutes were averaged across the recording period for each subject and multiplied by seven.For all analyses the statistical program R, version 3.2.0 , was use2). The final study population comprised of 475 subjects with a mean age of 58 years  for males and 28 minutes for females (25-75th percentile: 17–47 minutes) (p<0.05). In the total population, of the daily recorded time, a median of 61% (25-75th percentile: 54–67%) was spent in sedentary behavior, whereas the median time spent in light and MVPA was 35% (25-75th percentile: 30–42%) and 3% (25-75th percentile: 2–5%), respectively.The overall median recorded wear time averaged per subject was about 15 h/day . The daiAge comparisons revealed that subjects aged <55 years spent more minutes in MVPA/day than those aged >61 years, irrespective of sex . Normal and overweight men and women spent more time in MVPA/day compared to obese subjects ; Table C in Whilst in sedentary and light activity no associations were seen with age and BMI in the basic models, negative associations were present between MVPA and the highest age tertile  0.65, 0.87)) as well as obesity  . ExclusiIn our cohort, no or only unstable associations of morbidity with PA levels could be determined. Pain or physical complaints were negatively associated with MVPA in the best fit model obtained by stepwise selection, but were not significant after exclusion of subjects with myocardial infarction and those with stroke (p<0.1). Results were only slightly altered after exclusion of subjects who reported fair/poor overall health. Due to an interaction effect between sex and diabetes, we stratified the models of sedentary and light activity by sex. After stratification, diabetes showed a negative association with light activity and a positive association with time spent sedentary in females only p<0.05; . FurtherThe majority of time spent in MVPA was accumulated in short bouts i.e. less than 5 minutes . OverallWe found that 168 subjects in our population did not achieve a single episode of MVPA lasting at least 10 minutes. While 26% of normal weight subjects did not reach at least one episode of MVPA spent in at least 10 minutes, this amount was almost double in obese subjects (45%) . The highest prevalence of adherence was found within the middle aged group with 55 to 61 years (16%). Among subjects adhering to the WHO recommendation the prevalence of having hypertension, diabetes, asthma, COPD, walking difficulties, pain/physical complaints or anxiety/depression ranged from 3%  up to 24% (hypertension) , whereas group comparisons considering sex, age and BMI showed similar trends. Males, younger subjects and subjects with a lower BMI spent more time in MVPA  met the threshold of at least 2.5 hours of MVPA/week in bouts of at least 10 minutes.The low prevalence of adherence to the WHO PA recommendation is comparable to other European studies. In Norway, 20% of the study population (N = 3267), aged 20–85 years, accumulated at least 30 minutes of daily MVPA in bouts of 8–10 minutes . Of 1114In our analysis, in both, men and women, the majority of wear time was spent sedentary, being higher in males than in females. A meta-analysis has recently reported that the risk for all-cause mortality is increased for subjects with high amounts of sedentary behavior, assessed as sitting time, but that this effect seems to be mitigated through PA . SubjectGerman large scale population-based data, based on self-reports, reported an adherence to WHO PA recommendation of 18%, both in the 50–59 years and the 60–69 years age groups . This prOur data showed that the median number of daily minutes of MVPA decreased across age and BMI categories, and further, that males achieved more minutes of MVPA, which is in line with other studies , 13, 14.No stable association of hypertension, asthma, diabetes, COPD, walking difficulties, pain/physical complaints, or anxiety/depression with time spent in MVPA was found. This result should be interpreted with caution due to the low prevalence of morbidities such as COPD (9%) or diabetes (7%) and probably mainly mild disease stages in our study population. Despite the possibility that more active subjects participated in the PA assessment, our results indicate that there is an urgent need to improve PA among adults in southern Germany, given that subjects spent a median of 61% of the time sedentary, and in contrast only 3% in MVPA/day. Furthermore, only 14% met the WHO PA recommendation.The present results, including subjects aged 48–68 years resident in the region of Augsburg in southern Germany, are limited to the selected study population which had a relatively low response of 54%, likely leading to a potential risk of selection bias. Further, our results are based on cross-sectional data, which does not allow generalizations about long term PA behavior. Within our study population, the prevalence of morbidities was low and probably at early stages. Furthermore, these were assessed by questionnaires only and were not verified by an examination from a physician. The assessment of pain or physical complaints, and walking difficulties was based on self-report without precise definition of the cause.Despite these limitations, this study has several strengths revealing new insights into PA levels and duration pattern of German adults assessed by accelerometry. Data on objectively measured PA among German adults is rare. Despite the inability to measure all PA engagement equally well, the advantage of the use of accelerometers is the ability to analyze the intensity and duration of consecutive minutes of PA, while minimizing the risk of reporting bias occurring in self-reports , 9, 10.There is a lot of methodological diversity among studies investigating PA with accelerometers . This isIn conclusion, the prevalence of being physically active was found to be generally low among adults from southern Germany. Overall, time spent in MVPA was accumulated mainly in very short bouts and one third of the population did not achieve a single 10 minute bout of MVPA contributing towards the WHO PA recommendation. Furthermore, only one in eight subjects achieved the WHO threshold, indicating a further need for PA promotion in Germany, particularly among older and obese subjects.S1 Fig(PDF)Click here for additional data file.S1 Methods(PDF)Click here for additional data file.S1 Tables(PDF)Click here for additional data file.


The early detection of bladder cancer is important as the disease has a high rate of recurrence and progression. The development of accurate, non-invasive urinary assays would greatly facilitate detection. In previous studies, we have reported the discovery and initial validation of mRNA biomarkers that may be applicable in this context. In this study, we evaluated the diagnostic performance of proposed molecular signatures in an independent cohort.Forty-four mRNA transcripts were monitored blindly in urine samples obtained from a cohort of 196 subjects with known bladder disease status (89 with active BCa) using quantitative real-time PCR (RT-PCR). Statistical analyses defined associations of individual biomarkers with clinical data and the performance of predictive multivariate models was assessed using ROC curves. The majority of the candidate mRNA targets were confirmed as being associated with the presence of BCa over other clinical variables. Multivariate models identified an optimal 18-gene diagnostic signature that predicted the presence of BCa with a sensitivity of 85% and a specificity of 88% (AUC 0.935). Analysis of mRNA signatures in naturally micturated urine samples can provide valuable information for the evaluation of patients under investigation for BCa. Additional refinement and validation of promising multi-target signatures will support the development of accurate assays for the non-invasive detection and monitoring of BCa. Being among the five most common malignancies worldwide, bladder cancer (BCa) is a major cause of morbidity and mortality , 2. AlthIn previous studies , 5, we dA significant association with the presence of BCa was confirmed for the majority of our candidate diagnostic biomarkers , and forA panel of candidate mRNA biomarkers was monitored in an independent set of naturally voided urine samples. Twenty-one targets were selected from our previous discovery studies that identified a set of transcripts that were significantly associated with the presence of BCa , 5. An aP-value. The majority of the markers selected from urinary RNA-based, discovery and validation studies were confirmed as being significantly associated with the presence of BCa. Conversely, other than CTAG2, the biomarkers included from tissue-based studies [P <0.05) were significantly associated with the presence of gross hematuria, or gender  with es Florida . Of the The development of accurate assays for the non-invasive detection of bladder cancer continues to be a challenge. A number of tests have been developed to detect tumor-associated urinary biomarkers, but due to poor sensitivity and overall accuracy, none of these assays have sufficient predictive power to be applied to the management of individual patients . A shiftet al. identified a 12-gene signature that achieved good accuracy in diagnosing BCa in an independent cohort utilizing quantitative RT-PCR. In a validation study the 12-gene signature achieved a sensitivity of 80% with 86% specificity for discrimination of patients with BCa from controls [To look for BCa-associated mRNA signatures in bladder cancer, we previously applied a high-throughput molecular profiling and bioinformatics strategy to urothelial cell samples that are naturally shed from the bladder lining and can be readily recovered from urine . The ratcontrols , and an controls . Togethecontrols , 8, 23.This study represents an additional step towards the derivation of an accurate RNA-based diagnostic test for BCa detection. The selected targets were quantitated in an independent cohort that, although also collected at a Florida institution, represents a patient population distinct from that used in our discovery studies , 5. FurtP-values would be criteria for selecting the best biomarkers for further study, but the presence/absence rate in cases and control also provides valuable information. A target (e.g. SNAI2) that is absent in the majority of controls but present in the majority of cases would have good potential as a BCa detection biomarker. Conversely, a biomarker that is absent in both cases and controls may be less valuable (e.g. SEMA3D and CRH), even when differential expression was statistically significant. Another criterion for candidate biomarker selection can be the association with specific clinical variables. While association with age, gender or the presence of hematuria could negatively influence the inclusion of a biomarker for a broad spectrum BCa test, the inclusion of markers that are significantly associated with BCa plus stage or grade might provide additional information with regard to patient evaluation and management.Analyses confirmed that the majority (75%) of candidate biomarkers tested in the study cohort were strongly associated with the presence of BCa. Strong estimates and low In both univariate and multivariate analyses, the Florida panel provided the majority of the top-ranked biomarkers. This is to be expected given the number of candidates in each original panel and the fact that the study cohort in this study was similar to that used in our discovery studies , 5. The We recognize that the study has several limitations. Although both cases and controls were collected consecutively, we only initiated molecular analyses when the balance of samples in each group approached 50%. Disease prevalence is typically considerably lower in urologic practice, so evaluation of the validation study cohort is likely to provide an overly optimistic assessment of the assay predictive value. While this study represents an advance over previous studies that used only healthy controls , as a teWith an estimated 77,000 new cases in 2016 [g, 4°C, 5 min), rinsed in PBS, pelleted again, and frozen for storage at −80C. Total RNA was purified using Qiagen RNeasy kit with subsequent Qiagen DNase treatment. RNA samples were evaluated quantitatively and qualitatively using an Agilent Bioanalyzer 2000, before storage at −80°C as previously described [Under IRB approval and informed consent, urine samples and associated clinical information were consecutively collected from subjects visiting the urology clinic at MD Anderson Cancer Center at Orlando Health, Orlando, FL between 2011 and 2014. The study cohort consisted of 107 individuals with no evidence of active urothelial cell carcinoma (controls) and 89 individuals with newly diagnosed primary urothelial carcinoma (cases). All subjects underwent standard clinical work-up, including office cystoscopy, and the majority also had axial imaging of the abdomen and pelvis. For the bladder cancer case group, histological confirmation of urothelial carcinoma, including grade and stage was defined from excised tissue. A summary of clinical data is given in Table endogenous controls - PPIA, GAPDH, UBC, PGK1, identified previously [Florida Panel - BIRC5, ANG, CA9, AGT, DMBT1, ERBB2, CCNE2, SNAI2, MMP12, DSC2, TMEM45A, SYNGR1, MMP10, VEGFA, CCL18, SERPINE1, MMP1, MMP9, MXRA8, SEMA3D, and RAB1A. The gene expression assays for the targets described above were the same as those used in our previous studies [et al. in studies that also used isolated urothelia samples for bladder cancer detection [Barcelona Panel – TERT, KRT20, CRH, KFL9, MAGEA3, SLC1A6, POSTN, AHNAK2, ANXA10, CTSE, and PPP1R14D. The PCR primers for these targets were obtained from the associated articles [Australasia Panel - CDK1, MDK, IGFBP5, HOXA13 and CXCR2. Finally, we included a few additional targets gleaned either from bladder tumor tissue studies – PRAME, CTAG2 [Taqman low density arrays (TLDAs) were constructed by Applied Biosystems (AB). The TLDA format is a 384-well system that uses standard TaqMan assays and enables automated loading and high-throughput analyses . Targetseviously  using poetection , 8, herearticles , 8. A 5-E, CTAG2 , 12. PCRComplementary DNA was synthesized from 20 to 500 ng of total RNA, using the High Capacity cDNA Reverse Transcriptase Kit  following the manufacturer's instructions, with random primers in a total reaction volume of 20μl. A multiplex RT-PCR pre-amplification reaction was performed using the pooled 48 TaqMan Gene Expression Assays as described previously . The reaP<0.05 were deemed statistically significant.Differences in clinical covariates between bladder cancer cases and non-malignant controls were evaluated via Chi-squared test and Wilcoxon Rank Sum test, as appropriate. For each gene, the percentage of samples that were censored  was calculated for cases and controls separately. All four of the control genes had 100% observed data, that is, there were no Ct values <40. To determine the adequacy of each of these four genes as control genes, we used the t-statistic to compare cases and controls and observed that GAPDH and UBC were significantly associated with case-control status. Thus, only PGK1 and PPIA were used to normalize the 44 biomarkers of interest. To avoid biased inference caused by the issue of qPCR non-detects , we used a left-censoring approach . Ct valu


Acacia auriculiformis, being a halophyte, offers diverse benefits against soil salinity; however, the defense mechanisms underlying salt-tolerant capacity in A. auriculiformis are still elusive. In this study, we aimed to elucidate mechanisms regulating the adaptability of the multi-purpose perennial species A. auriculiformis to salt stress. The growth, ion homeostasis, osmoprotection, tissue tolerance and Na+ exclusion, and anatomical adjustments of A. auriculiformis grown in varied doses of seawater for 90 and 150 days were assessed. Results showed that diluted seawater caused notable reductions in the level of growth-related parameters, relative water content, stomatal conductance, photosynthetic pigments, proteins, and carbohydrates in dose- and time-dependent manners. However, the percent reduction of these parameters did not exceed 50% of those of control plants. Na+ contents in phyllodes and roots increased with increasing levels of salinity, whereas K+ contents and K+/Na+ ratio decreased significantly in comparison with control plants. A. auriculiformis retained more Na+ in the roots and maintained higher levels of K+, Ca2+ and Mg2+, and K+/Na+ ratio in phyllodes than roots through ion selective capacity. The contents of proline, total free amino acids, total sugars and reducing sugars significantly accumulated together with the levels of malondialdehyde and electrolyte leakage in the phyllodes, particularly at day 150th of salt treatment. Anatomical investigations revealed various anatomical changes in the tissues of phyllodes, stems and roots by salt stress, such as increase in the size of spongy parenchyma of phyllodes, endodermal thickness of stems and roots, and the diameter of root vascular bundle, relative to control counterparts. Furthermore, the estimated values for Na+ exclusion and tissue tolerance index suggested that A. auriculiformis efficiently adopted these two mechanisms to address higher salinity levels. Our results conclude that the adaptability of A. auriculiformis to salinity is closely associated with ion selectivity, increased accumulation of osmoprotectants, efficient Na+ retention in roots, anatomical adjustments, Na+ exclusion and tissue tolerance mechanisms.Salinity, one of the major environmental constraints, threatens soil health and consequently agricultural productivity worldwide.  Salinization of soil and groundwater has been considered as the most critical environmental issue, hindering sustainable agricultural productivity and presenting a challenging task for the environmentalists, especially in the era of global climate change. Salinity is a widespread problem, affecting around 831 million hectares of lands that include 397 and 434 million hectares of saline and sodic soils, respectively . It is u+ uptake by roots and/or increasing Na+ efflux back to the soil, intracellular Na+ sequestration, potassium retention in the cytosol, tissue-specific Na+ sequestration, control of ion loading in xylem and oxidative stress tolerance  that is native to Papua New Guinea, Northern Australia and Indonesia, which was brought for cultivation in Bangladesh in 1980s due to its fast growing nature, ensuring an adequate wood supply to sustain the country’s existing wood-based industries  plant growth and biomass, (ii) salt uptake and accumulation, (iii) mineral homeostasis, (iv) photosynthetic pigments (e.g. chlorophylls), (v) stomatal conductance, (vi) lipid peroxidation, (vii) osmoregulation, (viii) anatomical features of roots, stems and phyllodes, and (ix) salt exclusion and tissue tolerance index in response to increasing doses of external salinity.Therefore, in the present study, we aimed to examine the effects of a gradient of salinity on the growth and development of A. auriculiformis seedlings of uniform size were collected from Bangladesh Agricultural Development Corporation, Gazipur, Bangladesh, and each seedling was transplanted into each pot (26.5 cm in height and 27.5 cm in diameter). The pots were previously filled with soil mixture that was prepared by mixing oven-dried soil and cow dung (4:1). Each pot contained 12.04 kg of soil mixture and the soil moisture content was approximately 17% of field capacity. The soil was also treated with formaldehyde to curtail the probability of developing soil-borne diseases. In addition to cow dung, 1 L of liquid fertilizer containing urea, triple super phosphate and potassium sulfate (3% of each component) was applied to each pot at 30-day interval during the experimental period.A pot experiment was designed at the research field of the Department of Agroforestry and Environment, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh  for a period of 5 months (January 2014– June 2014). The study site is 8.2 m above the sea level and characterized by a sub-tropical climate with hot summer and mild winter. The minimum and maximum temperatures of the research area were fluctuated between 7–23°C and 28–39°C, respectively, during the experimental period. One-year-old -1, and Na+, K+, Ca2+ and Mg2+ concentrations of 478.70, 10.38, 9.90 and 56.08 mmol L-1, respectively) was diluted with tap water to formulate 4, 8 and 12 dS m-1 of salinity. For imposing salt stress, the uniformly grown plants were irrigated with the diluted seawater (300 mL) at 3-day interval for a period of 150 days. The control plants were irrigated with tap water devoid of seawater (0 dS m-1). Root, stem and phyllode samples were harvested at days 90th and 150th of salt treatments for determining various physiological and biochemical parameters. The experiment was conducted in randomized complete block design with five replicates in each treatment, and each replicate comprised one plant.After transplanting the seedlings into pots, the plants were allowed to grow for 10 days for adaptation prior to being treated with salt stress. Seawater collected from southern coastal area (Cox’s Bazar sea beach) of Bangladesh , respectively. For determining biomasses of phyllodes and roots, plants were carefully uprooted at day 150th of salt imposition and washed with distilled water to remove adherent soil. Dry weights (DWs) of phyllodes and roots were measured following the method described by Plant height and phyllode area were measured at days 90+, K+, Ca2+ and Mg2+. After desiccation, the powdered samples were used to determine the ion contents by an atomic absorption spectrophotometer  following the method of The oven-dried phyllode and root samples were ground into powder and kept in a desiccator for determining the contents of NaThe fresh, turgid and DWs of the phyllodes were used to determine phyllode relative water content (RWC) according to the formula developed by The levels of proline (Pro), total free amino acids and soluble proteins in phyllodes were determined following the methods described by Electrolyte leakage (EL) and the content of lipid peroxidation product [malondialdehyde (MDA)] in phyllodes were determined according to The contents of total soluble sugars and reducing sugars, and total carbohydrates in phyllode samples were colorimetrically estimated according to the method of Salinity-induced anatomical changes were examined by sectioning phyllode, stem, and root tissues under the light microscope  using a cleaned razor blade . The sections from control and seawater-treated plants were stained separately with safranin and fast-green following the method described by Salt tolerance index (STI) was calculated by using the following formula developed by where, TDW = total DW, SI = control treatment, Sx = salt treatment.Tissue tolerance was evaluated using the formula below according to 2, W2, (L+W)2 and the product L × W. The final model was selected based on the combination . The predicted area of natural senescence in salt-treated plants was calculated using the standard equation developed by regression analysis between projected phyllode area and senescenced phyllode area  was calculated by dividing the value of tissue tolerance at different doses by the value of the highest tissue tolerance obtained at any dose of salinity.+ exclusion was determined as the ratio of the Na+ concentration in the 4th phyllode and the Na+ concentration in the soil sample using the equation described by NaP < 0.05, according to least significant difference (LSD) test using Statistix 10 package. Data presented are means ± standard errors (SEs) of five replications (n = 5) for each treatment.The data were subjected to a one-way or two-way analysis of variance (ANOVA), and different letters indicate significant differences among treatments at A. auriculiformis, and the overall growth performance gradually declined upon increasing the level of salt stress for a period of 150 days . Salt stress caused a significant reduction of plant height by 15, 25 and 31% at day 90th, and 21, 30 and 39% at day 150th after exposure of plants to 4, 8 and 12 dS m-1 salinity levels, respectively, as compared with the respective control values (Table 1). Reduction of phyllode area progressively augmented with increasing salinity levels, and phyllode area was found to be reduced by 24, 44 and 54% at day 90th, and 39, 56 and 68% at day 150th in comparison with control after application of 4, 8 and 12 dS m-1 salinity levels to plants, respectively (Table 1).Salinity had considerable negative effects on the growth of A. auriculiformis were also assessed by determining DW of phyllodes and roots, DW-based phyllode/root ratio and root length after 150 days of salt treatment (Table 2). DW of salt-stressed phyllodes reduced by 26, 38 and 49%, while that of salt-treated roots by 31, 43 and 54% at 4, 8 and 12 dS m-1 salinity levels, respectively, compared with that of control. Consequently, DW-based phyllode/root ratio increased by 8, 8 and 12% at 4, 8 and 12 dS m-1 saline levels, respectively, in comparison with control. Careful evaluation of the length of roots showed that root length decreased by 22, 32 and 43% at 4, 8 and 12 dS m-1 saline levels, respectively, when compared with control (Table 2). STI, a reliable criterion for salt tolerance (Table 2). Nevertheless, STI decreased only by 31% despite that the level of salinity used was increased three times from 4 to 12 dS m-1. Seawater-induced salinity significantly affected the stomatal density in the adaxial part of the 4th phyllode and the number of stomata was proportionally declined with the levels of salinity (Table 2). Specifically, the stomata density in the phyllode of salt-stressed A. auriculiformis was decreased by 15, 27 and 47% at 4, 8 and 12 dS m-1 salinity levels, respectively, in comparison with that of salt-free control plants.The negative consequences of salinity on growth performance of olerance , was alsA. auriculiformis after salt treatment, and RWC was observed to reduce in a dose- and exposure time-dependent manner. In response to seawater-induced salinity, phyllode RWC decreased by 12, 19 and 29% at day 90th, and 26, 41 and 49% at day 150th under 4, 8 and 12 dS m-1 salinity levels, respectively, compared with control . The influence of salinity stress on stomatal conductance of A. auriculiformis plants  was similar to that on RWC. Stomatal conductance decreased in a salt concentration-dependent manner by 18, 36 and 45% at day 90th, and 30, 47 and 62% at day 150th of imposition of 4, 8 and 12 dS m-1 salinity, respectively, as compared with control. Our data also indicated that the stomatal conductance of the old plants were significantly higher than that of young plants under both normal and salt stress conditions.Phyllode RWC was determined at different stages of development of A. auriculiformis plants with diluted seawater caused gradual and significant decreases in the contents of Chl a , Chl b  and total Chls  following imposition of 4, 8 and 12 dS m-1 salinity, respectively, compared with plants irrigated with tap water (Table 1). Severe salt stress (12 dS m-1) exhibited a higher reduction in Chl a content (by 42%) than in Chl b (by 35%), leading to a conspicuous decrease in the overall Chl content (40%), when compared with control at day 150th of salt treatment. Additionally, the decreasing patterns of Chl a and Chl b also significantly altered Chl a/Chl b ratio in plants treated with diluted seawater (Table 1).Irrigation of A. auriculiformis . There were no significant changes in MDA contents at low salinity; whereas at moderate and high salinity (8 and 12 dS m-1), MDA content significantly increased by 6 and 30% at day 90th of salt treatment compared with control. On the other hand, at day 150th of salt imposition, MDA content significantly increased by 9 and 12% at moderate (8 dS m-1) and high (12 dS m-1) salinity levels, respectively, relative to control. There was also a sharp increase in the content of EL upon increasing the levels of salinity. Nevertheless, the increment of EL levels showed the maximum at the highest salinity dose (12 dS m-1), with 49 and 59% higher EL at days 90th and 150th, respectively, than that of untreated respective control.To assess the cell membrane integrity under salt stress, we determined the levels of lipid peroxidation product MDA and EL in the phyllodes of A. auriculiformis plants under normal and salt stress conditions. Pro content remarkably increased by 298, 430 and 583% at day 90th, and 265, 371 and 447% at day 150th after imposition of 4, 8 and 12 dS m-1 diluted seawater, respectively, compared with control . There was a gradual increase in the content of total free amino acids upon increasing the levels of salinity and exposure times . At day 150th of salt treatment, the levels of total free amino acids were elevated by 38, 54 and 116% at 4, 8, and 12 dS m-1 salinity levels, respectively, in comparison with control. Similar to the trend of total free amino acid contents, there was a positive relationship between salt concentrations and the levels of total soluble sugars and reducing sugars, and the maximum levels were noted at the highest level of salinity (12 dS m-1) at both days 90th and 150th of plant exposure to salt stress. At day 90th of salt stress, the level of total soluble sugars was enhanced by 4, 13 and 21% , and that of reducing sugars by 22, 48 and 109%  at 4, 8 and 12 dS m-1 salinity levels, respectively, compared with control. Upon increasing the salt-exposure time for an additional 60 days, the level of total sugars further increased by 22, 26 and 38%, and that of reducing sugars by 44, 84 and 131% at 4, 8 and 12 dS m-1 salinity levels, respectively, as compared with control . On the other hand, the contents of total carbohydrates and soluble proteins were negatively correlated with increasing doses of seawater . At day 90th of salt treatment, total carbohydrate content decreased by 14, 32 and 39%, while that of soluble proteins by 9, 19 and 28% at 4, 8 and 12 dS m-1 salinity levels, respectively, compared with control . The contents of these biomolecules were further declined at day 150th of salt exposure, and their values reached the lowest levels  at the highest concentration of salinity (12 dS m-1) .To understand the mechanisms associated with osmotic adjustment under salt stress, we have determined the levels of Pro, total free amino acids, total sugars, total carbohydrates, reducing sugars, and soluble proteins in A. auriculiformis, we measured the contents of Na+, K+, Ca2+ and Mg2+ in roots and phyllodes at day 150th of salt treatment. As shown in Figures 3A,B, treatment of plants with 4, 8 and 12 dS m-1 salinity levels resulted in a noticeable increase in the content of Na+ , whereas a significant reduction in K+ content  in salt-stressed plants compared with untreated control plants. It is worth mentioning that lower levels of Na+ and higher levels of K+ were accumulated in phyllodes, when compared with those in roots, indicating a higher salt tolerance tendency of A. auriculiformis. We also noted a significant decrease in the contents of Ca2+ and Mg2+ in both roots and phyllodes of salt-stressed plants, as compared with that in seawater-free control plants . The levels of Ca2+ and Mg2+ were also considerably higher in phyllodes than in roots of salt-stressed plants. We also calculated the K+/Na+ ratios, and found that salinity caused a decreasing trend of the ratios in both phyllodes and roots with progressive increase of salinity levels. However, it is worth mentioning that phyllodes displayed higher K+/Na+ ratios than roots . Thus, retention of Na+ in roots and maintenance of higher levels of K+, Ca2+ and Mg2+ in phyllodes enabled A. auriculiformis to adjust its ion homeostasis under salt stress.To get an insight into ion homeostasis mechanism of A. auriculiformis during salt stress, we analyzed the cross sections of phyllodes, stems and roots at day 150th of plant exposure to different levels of salinity . Our observations implied that the anatomical features of phyllodes, stems and roots significantly altered when A. auriculiformis plants were exposed to external salinity. In phyllodes, the thickness of upper and lower epidermises increased at low and moderate salinity levels (8 and 12 dS m-1) but decreased at high salinity (12 dS m-1), relative to control counterparts . Progressive increment of salinity levels triggered the loss of Chl substances in palisade parenchyma tissues. We also observed that the size of the spongy parenchymal layer gradually increased with increasing salinity levels. Salt stress resulted in a significant decrease in cortical area of stems of A. auriculiformis but no significant variation was found in case of epidermis even at the highest dose of salinity (12 dS m-1) . Endodermal thickness of stems was found to be increased with increasing salinity when compared with those of the stems of control plants . The vascular bundles of the stems enlarged at low (4 dS m-1) and moderate salinity (8 dS m-1); but decreases at high salinity level (12 dS m-1). On the other hand, salt-mediated toxicity increased the number of the cells in pith area of stems up to 8 dS m-1 salinity; thereafter, a decrease in number of cells was observed at 12 dS m-1. Results in Figure 4C indicated that the thicknesses of endodermis, pith area and the diameter of vascular bundles increased upon increasing the salinity levels . Interestingly, the cortical area in roots expanded at low intensity of salinity (4 dS m-1), then decreased at moderate and high levels of salinities (8 and 12 dS m-1). Moreover, salt stress triggered the increment of intercellular space and rupture of cell membrane of root tissues , which might be resulted from the formation of aerenchyma, a unique adaption feature of salt-affected tissues . The lowest and highest excluders of Na+ were given a value of 1 and 0, respectively, to develop a standardized Na+ exclusion index. According to this scale, exposure of plants to 4, 8 and 12 dS m-1 salinity exhibited a Na+ exclusion index of 0.91, 0.84 and 0.79, respectively, compared with control (1.0). The data presented in Table 4 exemplified that plants with low degree of salt-induced senescence and high Na+ concentrations were deemed to be tissue-tolerant. A measure for tissue tolerance, therefore, needs to consider the amount of Na+ accumulated in the phyllodes. Before calculating the TTI, it was necessary to evaluate natural phyllode senescence and necrosis in salt-stressed plants in order to determine the extent of Na+ prompted senescence. The index was scaled to a range between 0 and 1 by dividing the tolerance value of those salinity levels by the highest tolerance value. Thus, the highest tissue tolerance would have a value of 1, while the theoretically lowest tissue tolerance would have a value of 0. Our data analysis indicated that A. auriculiformis had the highest TTI (1) at 4 dS m-1 salinity followed by 8 dS m-1 (0.91) and 12 dS m-1 (0.74) salinity levels.At day 150A. auriculiformis by interfering various parameters upon which its growth relies . We observed a salt-induced increasing trend of phyllode/root ratio (Table 2), which was supported by the findings of Figure 5).One of the general consequences of salt toxicity is to inhibit plant growth, resulting in the generation of stunted plants. At above the threshold level of salt, the growth rate and biomass of all plants progressively decrease, although the degree of damage varies among plant species . In the a and Chl b, are vital for steady photosynthesis in plants during salt stress. It has been reported that photosynthesis in some halophytes remains unaffected by salinity or even increases at low salinity . The reduction in Chl levels and consequent biomass loss were evident in other studies under salt stress, and the possible cause was attributed to the direct effects of accumulated Na+ and ROS , as was repoted in Atriplex helimus by Bendaly et al. (2016)Photosynthetic efficiency determines the overall performance of plants, which is reflected by growth and biomass parameters. Under salt stress, phyllode Chl content could be altered due to impaired biosynthesis and accelerated degradation of the pigments . Therefosalinity . In the  and ROS . In accoan Chl b . Chl a ihe Chl a . Thus, aA. auriculiformis was coincided with decreases in RWC, stomatal density and conductance, and membrane degradation . In this study, we observed an incremental reduction of phyllode RWC with increasing the concentration and exposure period of seawater, indicating salt-mediated induction of physiological water deficit in A. auriculiformis . Decreases in stomata number and conductance in phyllodes might be an adaptation mechanism that A. auriculiformis employs to optimize water use efficiency and to make spaces for sequestrating more Na+ under salt stress, as the similar tendency was also observed in other halophytes, such as Chenopodium quinoa  might be attributed to disparity of total stomatal number, stomatal morphology and number of chloroplasts, which is commonly found in the fully expanded leaves .Salt-induced decline in photosynthetic pigments in m quinoa . On the d leaves . Fontana+ initiates cellular toxicity by targeting cell membrane through the production of ROS. ROS mainly attacks the membrane, causing lipid peroxidation that leads to the generation of MDA and loss of essential electrolytes (-1); however, these two determinants drastically increased at severe salinity (12 dS m-1) and long-exposure period (150 days), indicating the capability of A. auriculiformis in resisting salt-induced membrane damage up to a certain level of salinity . These data also demonstrated the correlation between the significant increase of MDA content and EL, and the growth inhibition and metabolic disturbance under severe salt stress , as previously reported in other species . Pro is known to provide improved protection against salinity by scavenging free radicals, stabilizing membranes, proteins and enzymes, and maintaining ion homeostasis . Interestingly, the observed declines in total carbohydrate and protein contents endorsed the increased levels of sugars and amino acids , as well as enhanced rate of respiration to produce much ATP required for the activity of stressed cells  Na+ extrusion and (iii) sequestration of Na+ in the vacuoles . Additionally, impact of salt stress on the uptake and transport of other minerals is also considered important determinant of salt tolerance mechanism in plants . These data indicate that competitive inhibition between Na+ and K+ absorptions resulted in a change in the balance of intracellular K+/Na+ ratio in acacia plants. However, Na+ concentrations in roots were significantly higher than that of phyllodes , implying greater deposition of Na+ in the roots and reduced translocation of excessive Na+ from roots to aerial parts of acacia plants. This regulation of Na+ and K+ homeostases also implies the ability of A. auriculiformis to maintain a sufficient uptake of K+ in order to maintain a high cytosolic K+/Na+ ratio , which is one of the key determinants of plant salt tolerance  also indicated that ion exclusion mechanisms reduced the antagonistic effects of Na+, hence accelerating the uptake of other beneficiary nutrients. K+ actively participates in activating enzymes, stabilizing protein synthesis, maintaining membrane potential and cytosolic pH, whereas Ca2+ plays pivotal roles in K+/Na+ selectivity and signal transduction, and Mg2+ regulates cellular pH and ionic balance in addition to its central role in pigment formation .The salt tolerance in plants is invariably associated with three major components, including (i) Nan plants . In the olerance . The sigormation . Our res+ in salt-affected phyllodes . However, reduced epidermal thickness at high salinity indicated limited cell division and growth response under extreme salinity. The decrease in vascular tissue area under excessive salinity suggested an adaptation strategy to reduce water loss, which is supported by the findings of Salt stress also exerted severe load on the cellular organelles, and plants are accustomed to adjust the anatomical features in order to minimize the damage in presence of excessive NaCl. Phyllode anatomical observation showed increased thickness of upper and lower epidermises up to moderate salinity, indicating improved water use efficiency and sequestration of Nahyllodes  . The decrease in cortical area of stems might be a defensive strategy plants adapted to curtail growth under salinity by conserving essential energy for survival .The anatomy of stems was also severely altered upon imposing various doses of saline water , thereby suggesting a reduced diffusion of Na+ to the roots . Blackish spots and ruptured cells were also observed under elevated salinity , which attributed to the formation of ROS and MDA  accumulation of compatible solutes like Pro, total sugars, reducing sugars and total free amino acids; (ii) increase amount of K+, Ca2+ and Mg2+ in phyllodes than roots; (iii) increase K+ retention in photosynthetic tissues through hindering Na+ uptake; (iv) anatomical adjustment by increasing the size of spongy parenchymal tissue of phyllodes, endodermal thickness of stems and roots, and pith area of roots; (v) efficient Na+ sequestration in vacuoles that would be facilitated by a decrease in stomatal density and (vi) the enhanced Na+ exclusion and TTI. Our findings support that A. auriculiformis is a suitable perennial tree species to be grown for conservation of soil and ecology, as well as earning income in salt-affected areas owing to its popular use as fuel wood and materials for making paper and furniture.MMR, MAR, and SS conceived an designed the experiments. MMR, MAR, and SS conducted experiments. MMR and MGM analyzed and interpreted the data and results. MMR and MGM wrote and revised the manuscript. MGUM and MAK provided materials, monitored the experimental work and critically commented on the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.