Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms  in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples. Alpha-2-antiplasmin (α2AP), a member of the serine protease inhibitor (SERPIN) superfamily, is the main natural inhibitor of the fibrinolytic enzyme plasmin –3. NativIn the circulation α2AP undergoes both N-and C-terminal modifications, which significantly alter its inhibitory activity. In approximately 65% of circulating α2AP the C-terminus is intact so it can bind to plasmin(ogen) , whereas the remainder (~35%) has lost its ability to bind plasminogen  . ApproxiSERPINF2 gene, the gene coding for human α2AP, has been described previously [et al. showed that the Arg to Trp polymorphism of codon 6 (R6W) in the N-terminus of α2AP is functionally significant, as it affects the conversion of Met-α2AP to Asn-α2AP by APCE, and thereby the rate of α2AP incorporation into fibrin [A genetic variation in the eviously –16. Chrio fibrin . They shTo date, assays to specifically determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that can only measure Met(R6)-α2AP, as we previously described . The polα2AP-depleted plasma was obtained from Affinity Biologicals .Normal pooled plasma was prepared from citrated apheresis plasma  from 5 healthy donors. Pooled plasma from R6 homozygote individuals (R6 pooled plasma) or W6 homozygote individuals (W6 pooled plasma) was prepared by pooling 100 μl plasma samples from 18 and 17 previously genotyped control subjects of the ATTAC study population, respectively . GenotypGeneration of recombinant Met(R6)-α2AP and recombinant Asn-α2AP was performed as described previously . The wilmAbs against α2AP were produced in-house as described by Galfré and Milstein . A mixtuAll mouse experiments were carried out after prospective approval of the KU Leuven Ethical Committee for Animal Experimentation (ECD) . Anesthesia of the mice was performed using sevoflurane.2HPO4 and 1 mM KH2PO4 (pH 7.4)) of recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP for 24 hours at 4°C. Non-specific sites were blocked with 200 μl 1% bovine serum albumin  in PBS and incubated for two hours at RT. The plates were washed four times with 200 μl 0.002% Tween-80 in PBS (PBST). Subsequently, 180 μl of serial dilutions (100–0.0001 μg/ml) of purified mAbs in PBST containing 0.1% BSA were added to the wells and incubated for two hours at RT. After four rounds of washing with 200 μl PBST, HRP-conjugated polyclonal rabbit anti-mouse IgG (2 mg/ml) (Sigma) was added at an optimized dilution of 1:10000 and incubated for two hours at RT. Following four washes with 200 μl PBST, the enzymatic activity was determined using o-phenylenediamine  in a solution containing 0.1 M sodium citrate and 0.2 M di-sodium phosphate and H2O2  and the reaction was stopped after 45 minutes by adding 50 μl of 4 M H2SO4. The absorbance was read at 492 nm on a ELx808 Absorbance Microplate Reader .Purified mAbs were tested for their specific reactivities with α2AP by one-site non-competitive ELISA. 96-well microtiter plates  were coated with 2 μg/ml -α2AP or Met(R6)-α2AP were calculated by dividing the mAb concentrations at which half-maximal mAb binding occurred and by multiplying this ratio by 100.® 800CW donkey anti-mouse secondary antibodies (1 mg/ml)  were added at an optimized dilution of 1:10000 and incubated for one hour at RT. Following three washes with PBS, the blots were scanned in the 800 nm channel of an Odyssey® Imaging System .To investigate the reactivities of the mAbs by Western blot, recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP in TBS were boiled for five to ten minutes at 95°C in the presence of XT Sample Buffer and XT reducing agent  and resolved on a 10% SDS-polyacrylamide gel (300 ng/lane). After electrophoresis the proteins were transferred to a nitrocellulose membrane using a PowerPac™ HC power supply (BioRad) with transfer buffer  and 20% methanol) at 100V constant voltage for one hour. After protein transfer, non-specific sites on the nitrocellulose membrane were blocked with 5% milk in PBS, pH 7.4 followed by three washings in PBS with 0.1% Tween-20. Post-blocking, the blots were incubated with selected purified mAbs (800 ng/ml) in PBS with 0.1% Tween-20 for 18 hours under constant motion at 4°C. After three washes with PBS with 0.1% Tween-20, IRDye5,5′-tetramethylbenzidine (Sigma) as substrate, and the reaction was stopped after ten minutes by adding 100 μl per well of 1 M H2SO4. The absorbance was read at 450 nm on a Victor3 1420 multilabel plate counter .Three selected mAbs (MA-AP37E2 for Met(R6)-α2AP, MA-AP34C4 for Met(W6)-α2AP and MA-AP15D7 for all α2AP variants ) were used to develop sandwich-type ELISAs. MA-AP15D7 was HRP-conjugated to function as detection antibody. 96-well microtiter plates  were coated with 2 μg/ml of MA-AP37E2 or MA-AP34C4 (110 μl per well in 0.05 M carbonate buffer (pH 9.6)) overnight at 4°C. Non-specific sites were blocked with 150 μl 1% BSA in PBST for one hour at RT and plates were washed once with PBST. Subsequently, serial dilutions (1000–15.6 ng/ml) of recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP with 0,1% BSA in PBST were added. Plates were incubated for two hours at RT followed by four rounds of washing with PBST. Next, MA-AP15D7-HRP (2 mg/ml) was added and incubated for one hour at RT. For the Met(R6)-α2AP ELISA MA-AP15D7-HRP was diluted 8000 times and for the Met(W6)-α2AP ELISA MA-AP15D7-HRP was diluted 2000 times. Following four washes with PBST, enzyme activity was determined using 3,3′,For the analysis of native Met-α2AP in plasma, serial dilutions of plasma samples  were added. The assays were performed using plasma dilutions of 1:40–1:2560 for the Met(R6)-α2AP ELISA and plasma dilutions of 1:5–1:320 for the Met(W6)-α2AP ELISA. The responses of the different plasma samples in the ELISAs are expressed as the mean result of triplicate measurements.For the calculation of Met(R6)-α2AP and Met(W6)-α2AP concentrations in plasma samples we took the following in account: (1) normal pooled plasma of healthy individuals contains 70 μg/ml α2AP , which iVariation of Met(R6)-α2AP and Met(W6)-α2AP in R6 homozygote individuals (n = 18) and W6 homozygote individuals (n = 17) was determined by the Met(R6)-α2AP ELISA and Met(W6)-α2AP ELISA, respectively. The assays were performed using plasma dilutions of 1:600 for the Met(R6)-α2AP ELISA and plasma dilutions of 1:20 for the Met(W6)-α2AP ELISA. Normal pooled plasma was used to determine the inter- and intra-assay coefficients of variation (CV) of the ELISAs.The conversion of Met-α2AP into Asn-α2AP by APCE was assessed by incubating normal pooled plasma at 29°C for 0, 6, 24, 30, 48, 54 and 72 hours followed by analysis in the Met(R6)-α2AP and Met(W6)-α2AP ELISAs.In total a panel of 25 mAbs against α2AP was generated of which 8 mAbs were specific for Met-α2AP. For the development of novel Met-α2AP ELISAs we selected three mAbs. MA-AP37E2 and MA-AP34C4 were selected for their relatively specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP respectively . MA-AP15MA-AP37E2 showed 0.1% cross-reactivity with Met(W6)-α2AP and MA-AP34C4 showed 0.01% cross-reactivity with Met(R6)-α2AP . MA-AP37In addition to the ELISA analyses, we analyzed the reactivity of the selected mAbs by Western blot. Results showed reactivities of MA-AP37E2, MA-AP34C4 and MA-AP15D7 with protein bands with molecular weights of ~53-kDa, representing recombinant Met(R6)-α2AP, recombinant Met(W6)-α2AP and recombinant Asn-α2AP respectively ; S1 Fig.For the development of sandwich-type ELISAs, MA-AP37E2 (used in the Met(R6)-α2AP ELISA) and MA-AP34C4 (used in the Met(W6)-α2AP ELISA) were applied as coating antibodies and HRP-conjugated MA-AP15D7 as detection antibody. Recombinant Met(R6)-α2AP showed high reactivity in the Met(R6)-α2AP ELISA, whereas virtually no reactivity was observed for recombinant Met(W6)-α2AP and recombinant Asn-α2AP up to a concentration of 1000 ng/ml . RecombiApplying plasma to the novel ELISAs revealed a high reactivity for R6 pooled plasma in the Met(R6)-α2AP ELISA, but no reactivity for W6 pooled plasma. Additionally, W6 pooled plasma showed high reactivity in the Met(W6)-α2AP ELISA, but no significant response was displayed for R6 pooled plasma . A matriUsing the Met(R6)-α2AP and Met(W6)-α2AP ELISAs, we quantified native Met(R6)-α2AP and Met(W6)-α2AP in normal pooled plasma. We found that normal pooled plasma used in the current study contains 6.4 μg/ml Met(R6)-α2AP and 6.9 μg/ml Met(W6)-α2AP.Plasma samples of R6 and W6 homozygote individuals were analyzed in the Met(R6)-α2AP and Met(W6)-α2AP ELISA, respectively . Met(R6)Christiansen et al. showed in a purified system that APCE cleaves Met(R6)-α2AP approximately 8 times more quickly than Met(W6)-α2AP . To studCurrent assays to determine N-terminal variation of α2AP in plasma are limited to an ELISA that can only measure Met(R6)-α2AP . Here wePreviously, Christiansen et al. showed that R6 and W6 homozygous individuals have on average 23.6% Met(R6)-α2AP and 56.4% Met(W6)-α2AP, respectively . Based oEarlier studies have generated and characterized mAbs reacting with distinct parts of the α2AP protein. A mAb against α2AP’s C-terminus has been shown to be useful for the assessment of C-terminal cleavage , whereasIn a previous study on the assessment of α2AP N-terminal variation in plasma, the Met-α2AP ELISA used a polyclonal antibody raised against the first twelve N-terminal amino acids of Met-(R6)-α2AP . This poWe used the newly developed ELISAs to show that endogenous Met(R6)-α2AP present in plasma is converted more quickly into Asn-α2AP by APCE compared to Met(W6)-α2AP. This confirms and extends a previous study performed with purified Met(R6)-α2AP and Met(W6)-α2AP by Christiansen et al., who showed that Met(R6)-α2AP is a better substrate for APCE than Met(W6)-α2AP, since Met(R6)-α2AP was cleaved ~8 times more quickly to Asn-α2AP . PossiblThe selective reactivities of MA-AP37E2 and MA-AP34C4 in ELISA were not observed in the Western blot experiments. On the blots we found considerable cross-reactivity from both mAbs. The discrepancy between the ELISA- and Western blot results may partially be explained by our definition of cross-reactivity in ELISA -α2AP and Met(W6)-α2AP in plasma. This will enable us to improve the evaluation of the fibrinolytic potential in plasma samples in various disease states.S1 FigOriginal Western blot result showing the reactivity of (A) MA-AP37E2, (B) MA-AP34C4 and (C) MA-AP15D7 with recombinant α2AP. Lane 1: Met(R6)-α2AP (300 ng), lane 2: Met(W6)-α2AP (300 ng), lane 3: Asn-α2AP (300 ng). α2AP protein bands are visualized in green. The molecular weight marker proteins are visualized in red.(TIF)Click here for additional data file.


The risk of postoperative surgical site infection after long bone fracture fixation can be decreased with appropriate antibiotic use. However, there is no agreement on the superiority of a single- or multiple-dose perioperative regimen of antibiotic prophylaxis. The purpose of this study is to determine the following: 1) What are the current practice patterns of orthopaedic trauma surgeons in using perioperative antibiotics for closed long bone fractures? 2) What is the current knowledge of published antibiotic prophylaxis guidelines among orthopaedic trauma surgeons? 3) Are orthopaedic surgeons willing to change their current practices?A questionnaire was distributed via email between September and December 2015 to 955 Orthopaedic Trauma Association members, of whom 297 (31%) responded.Most surgeons (96%) use cefazolin as first-line infection prophylaxis. Fifty-nine percent used a multiple-dose antibiotic regimen, 39% used a single-dose regimen, and 2% varied this decision according to patient factors. Thirty-six percent said they were unfamiliar with Centers for Disease Control and Prevention (CDC) antibiotic prophylaxis guidelines; only 30% were able to select the correct CDC recommendation from a multiple-choice list. However, 44% of surgeons said they followed CDC recommendations. Fifty-six percent answered that a single-dose antibiotic prophylaxis regimen was not inferior to a multiple-dose regimen. If a level-I study comparing a single preoperative dose versus multiple perioperative antibiotic dosing regimen for treatment of closed long bone fractures were published, most respondents (64%) said they would fully follow these guidelines, and 22% said they would partially change their practice to follow these guidelines.There is heterogeneity in the use of single- versus multiple-dose antibiotic prophylaxis for surgical repair of closed long bone fractures. Many surgeons were unsure of current evidence-based recommendations regarding perioperative antibiotic use. Most respondents indicated they would be receptive to high-level evidence regarding the single- versus multiple-dose perioperative prophylactic antibiotics for the treatment of closed long bone fractures.The online version of this article (doi:10.1186/s13037-016-0118-5) contains supplementary material, which is available to authorized users. Surgical site infections (SSIs) account for approximately 38% of all postoperative infections and can be devastating after treatment of long bone fractures . Rates oAntibiotic administration before surgical incision has markedly improved the safety of modern surgery, especially in cases with high risk of infection, including the use of retained prosthetic instrumentation as is used in closed long bone fracture repair . AlthougWe hypothesized that there would be heterogeneity in practice patterns among orthopaedic trauma surgeons in terms of using single- versus multiple-dose antibiotic regimens. We also hypothesized that many orthopaedic trauma surgeons would be unfamiliar with published antibiotic prophylaxis guidelines but would be willing to change their practices to help prevent SSI. This study assessed the following questions: 1) What are the current practice patterns of members of the Orthopaedic Trauma Association (OTA) in using perioperative antibiotics for closed long bone fractures? 2) What is the current knowledge of published antibiotic prophylaxis guidelines among orthopaedic trauma surgeons? 3) Are orthopaedic surgeons willing to change their current antibiotic prophylaxis practices?After approval from our institutional review board and the OTA, an electronic survey  to summarize the responses to all survey questions and present descriptive statistics.A total of 297/955 (31%) members of the OTA completed the survey. Three incomplete surveys were received and were not included in the results of this study. Of the respondents, 21% had 0 to 5 years of post-training clinical practice, 23% had 6 to 10 years, 13% had 11 to 15 years, 19% had 16 to 20 years, 11% had 21 to 25 years, 9.1% had 26 to 30 years, and 6.1% had ≥31 years. The median number of long bone fractures treated annually per surgeon was 180 .n = 284) used cefazolin as their preferred first-line prophylaxis antibiotic for treatment of closed long bone fractures (Table n = 171) administered one dose of antibiotic within 1 h prior to incision and re-administered for 24 h (Table n = 56) administered one dose of antibiotic within 30 min prior to incision and did not re-administer postoperatively. Others  administered one dose within 1 h prior to incision and did not re-administer postoperatively. Others’ practices varied situationally, with 2% (n = 6) of respondents reporting varying their practices according to injury severity, fracture type, and operative intervention.Most respondents  said they re-administered every 4 h. Others re-administered every 3 h , 5 h , or 6 h . Ten percent of respondents (n = 30) said they did not re-administer antibiotics during surgery. Others (n = 7) said they re-administered antibiotics on the basis of estimated blood loss or antibiotic half-life.When asked how frequently they re-administer antibiotics during a procedure, 63% of respondents (n = 198) re-administered according to the half-life of the antibiotic or duration of surgery, and 20% (n = 54) re-administered according to estimated blood loss or intravenous fluid dilution as the case progressed. Others cited literature to support their dosing patterns or re-administered according to hospital guidelines, wound size, procedure type, or complexity of the case. Only three respondents (1.1%) re-administered before making a second incision in more complex surgical cases , 75% (n = 165) of respondents answered false, 5.4% (n = 16) answered true, and 39% (n = 116) were unsure. When asked if a multiple-dose antibiotic regimen  significantly reduced the risk of SSIs compared with a single dose of preoperative antibiotics, 62% (n = 185) of respondents answered false, 8.8% (n = 26) answered true, and 29% (n = 86) were unsure.When asked if a single-dose of long-acting intravenous antibiotic was inferior to any multiple-dose antibiotic regimen to prevent orthopaedic SSIs, 56% (n = 15) of respondents answered every 2 to 3 h, 36% (n = 106) answered every 4 to 6 h, 1.7% (n = 5) answered every 6 to 12 h, 26% (n = 77) answered when the duration of the procedure exceeds 1 to 2 times the half-life of the antibiotic, 2.7% (n = 8) answered when the duration of the procedure exceeds 3 to 4 times the half-life of the antibiotic, and 29% (n = 85) were unsure.When asked how frequently prophylactic antibiotics should be administered intraoperatively to maintain minimum inhibitory concentration, 5.1%  answered correctly: “maintain therapeutic levels of the antibiotic agent in serum and soft tissue throughout the operation and until, at most, a few hours after the incision was closed in the operating room.” Another 2.7% (n = 8) answered “maintain therapeutic levels of the antibiotic agent in serum and soft tissues throughout the operation and until 12 h after the incision is closed in the operating room”; 31% (n = 92) answered “maintain therapeutic levels of the antibiotic agent in serum and soft tissues throughout the operation and until 24 h after the incision is closed in the operating room”; 0% (n = 0) answered “maintain therapeutic levels of the antibiotic agent in serum and soft tissues throughout the operation and until 48 h after the incision is closed in the operating room”; and 36% (n = 107) were unsure of the published guideline. When asked if they follow this CDC guideline to prevent SSI, 44% (n = 132) said yes, 8.8% (n = 26) said no, 41% (n = 123) answered that they did not know the CDC-recommended dosing, and 5.4% (n = 16) preferred not to answer.When surgeons were asked which answer choice had category IA evidence to support its recommendation regarding perioperative antibiotic usage and dosing based on the  in 1999 , 30%  said they would fully follow these guidelines. Sixty-five respondents (22%) said they would somewhat follow the evidence but adjust dosing on a case-by-case basis; 7 respondents (2.4%) said they would continue their current practices, 19 respondents (6.4%) said they would follow their hospital’s guidelines rather than published guidelines, and 15 respondents (5.1%) said they were unsure if they would make a change to their current practices.We found heterogeneity in the use of single- versus multiple-dose antibiotics by orthopaedic trauma surgeons treating closed long bone fractures, with 39% administering only 1 preoperative dose and 59% using at least 1 additional postoperative dose. We also found that many respondents were unfamiliar with published antibiotic prophylaxis guidelines. Despite familiarity with recommendations that show no benefit of postoperative antibiotic prophylaxis, many surgeons continue to administer antibiotics postoperatively. Respondents said they would be willing to change their practices if a well-performed study were published regarding the use of perioperative antibiotics in closed orthopaedic trauma cases.Clinical practice guidelines for perioperative antibiotic prophylaxis developed jointly by the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, the Surgical Infection Society, and the Society for Healthcare Epidemiology of America recommend routine use of cefazolin unless contraindicated for “clean” orthopaedic procedures involving internal fixation such as the treatment of closed long bone fractures . In the The CDC’s Infection Control and Hospital Epidemiology guidelines published in 1999 recommend that antibiotics be re-administered if the duration of surgery is expected to exceed the time during which therapeutic levels of the antibiotic can be maintained and, at most, until a few hours after the surgery has ended  and metaTo our knowledge no high-level study has defined guidelines specific to orthopaedic closed long bone fractures. Accordingly, 64% of respondents were in favor of following recommendations that would be supported by a level-I study comparing a single-dose preoperative antibiotic with a multiple-dose perioperative antibiotic regimen. Twenty-two percent said they would “somewhat” change their practice, and only 2.4% said they would not change their practice if such guidelines became available.This study is limited by factors inherent in the survey methodology, including the risk of nonresponder bias. Certain questions designed to assess respondents’ knowledge of the orthopaedic literature in a true-or-false format may not capture respondents’ actual knowledge of the current orthopaedic literature regarding these topics. There may be self-reporting bias, with respondents possibly answering in a way that is a positive reflection of their practice, and it is difficult to control for respondents looking up answers and references before responding to questions in an online survey. However, respondents were asked to answer survey questions as objectively as possible, and the answer choice “unsure” was available and selected frequently, indicating that many respondents answered according to their current knowledge of the literature. It is difficult to determine whether the demographic characteristics and practice patterns of the responders to this survey reflect those of the orthopaedic community at large. Specifically, practice patterns of members of the OTA may not reflect practice variation within the orthopaedic community. This study has several strengths. First, only members of the OTA who treat primarily fractures were surveyed. Second, the response rate of 31% was high for a survey of the OTA, in which response rates are typically 10 to 15% –25. ThirOur study shows that there is heterogeneity in the use of antibiotic prophylaxis for surgical treatment of closed long bone fractures in terms of single- versus multiple-dose regimens. These variations likely arise from an experience-based approach, given that many survey respondents were unsure of current evidence-based recommendations regarding perioperative antibiotic use. The results indicate that the orthopaedic community would be receptive to high-level evidence regarding the efficacy of a single-dose versus multiple-dose perioperative antibiotic prophylaxis regimen for the treatment of closed long bone fractures. Studies focusing on this comparison are needed.


In the last decades, fastening systems have become an essential part of the construction industry. Post-installed mechanical anchors are frequently used in concrete members to connect them with other load bearing structural members, or to attach appliances. Their performance is limited by the concrete related failure modes which are highly influenced by the concrete mix design. This paper aims at investigating the effect that different aggregates used in the concrete mix have on the capacity of an undercut anchor under tensile quasi-static loading. Three concrete batches were cast utilising three different aggregate types. For two concrete ages (28 and 70 days), anchor tensile capacity and concrete properties were obtained. Concrete compressive strength, fracture energy and elastic modulus are used to normalize and compare the undercut anchor concrete tensile capacity employing some of the most widely used prediction models. For a more insightful comparison, a statistical method that yields also scatter information is introduced. Finally, the height and shape of the concrete cones are compared by highly precise and objective photogrammetric means. Compared to cast-in connection details, post-installed anchors provide the opportunity to design modular structures, accelerate the construction process and renovate or strengthen old buildings. In consequence, the usage of post-installed fastening systems has been increasing steadily during the last decades. Depending on the specific anchor system and load situation, several different failure mechanisms ranging from yielding of steel, failure of the adhesive interface or progressive fracture of the concrete substrate, leading to the so called “concrete cone break-out”, can occur. For the latter, several equations  havCast-in, as well as post-installed anchors, have been studied extensively with experimental and numerical means to identify the complex failure mechanism and its influencing factors, among them the concrete properties, the anchor geometry, and test configuration. Regarding the concrete properties, many studies have been performed showing differences in terms of anchor capacity for normal and high strength concrete . Also, eSince fasteners are known to attract or induce cracks, the behaviour of pre- and post-installed anchors have been studied extensively both in plain and cracked concrete . ConcreAnother influencing factor is the anchor geometry which has been studied in order to relate to the anchor’s features, not only the peak load, but also the global behaviour. For instance, Nilforoush et al. analysed the influence of the headed stud head size and the concrete slab thickness. They related their experimental results to the predictions obtained by equations ,10. In tWith the rise of computational power increasingly more important insights are gained by simulations. Artificial Neural Networks (ANN) have been successfully used to predict the capacity of anchors loaded in tension  or shearFinally, also the influence of the concrete properties on the anchor capacity has been numerically analysed . CompresIn order to maximise consistency between laboratories, and cover all relevant cases, specific guidelines were created ,24,25,26The concrete cone capacity is influenced by many parameters already under quasi-static conditions, not considering further loading and deterioration scenarios. For this reason, three concrete batches were cast utilising different aggregates but maintaining the overall mix design in an attempt to isolate a potential “aggregate effect” on the concrete cone failure of an undercut anchor product found on the market, as reported earlier . All theThis comprehensive dataset allows the direct investigation of correlations between material properties and system response. Furthermore, this data can also serve for the calibration of material parameters in numerical investigations which will be used to gain more insights into the problem, and/or extend numerically the experimental dataset (as done in ).After a short review of the concrete cone capacity design (CCD) method, the test results of the three different batches are systematically analysed and compared in terms of (a) depth and shape of the crater left in the slab after ripping out the concrete cone, and (b) the undercut anchor capacity in tension normalized by the concrete properties. The link between available concrete material properties and system response is studied utilising the currently established analytical prediction models.This manuscript deals with an undercut anchor product which is placed in unreinforced concrete and tested under pure tensile load in a configuration where the anchor response at peak load is not affected by the supports. In the fastening community, this set-up is referred to as “unconfined configuration”. In general, pull-out tests can be considered to be unconfined (see ) when thOther failure mechanisms in tension, like steel failure and pull-through failure , are relDuring the past years, solid design methods have been developed for fastening systems. Fuchs et al. introduced a design procedure, the concrete capacity design approach , which Equation  is basedies (see ). Comparons see ,34) and  and Pd ions .As just discussed, aggregate type, shape and size influence the concrete behaviour. Thus, an influence on the system behaviour can also be expected. The influence that the aggregates can have on the undercut anchor tensile capacity can be divided into two categories: (a) the direct influence on the concrete properties and consequently on the system behaviour; and (b) an indirect influence of the aggregates on the system behaviour that is not related to changes in concrete properties.In the second case, different aggregates would assert further influence on the system capacity and this influence would depend on the working principle of the considered anchor type. Typical working principles of anchors are interlock, friction and adhesion. In the specific case of the investigated undercut anchor product, the working principle is primarily interlock which is obtained by creating an undercut with a special drill bit. The drilling procedure and resulting undercut can be influenced by the aggregate type. Due to the heterogeneity of concrete, a drilled hole or undercut will rarely be perfectly axisymmetric, leading to a certain degree of uncertainty on the undercut geometry and on the resulting embedment depth. After the borehole is drilled, the anchor is installed and pre-tensioned. In this process the anchor is forced to adapt its shape to the previously generated undercut, establishing the actual mechanical interlock and embedment depth. Said uncertainty is addressed in The experimental campaign includes tests for the characterisation of the concrete material properties and unconfined pull-out tests on the investigated undercut anchor product in order to determine the anchor capacity in tension. Additionally, the used coarse aggregates were mechanically characterised by means of micro-indentation. For each of the three batches, three repetitions for each standard specimen  were tested, and five pull-out tests were performed. Slabs of 1.5 × 3.0 × 0.3 m were used for the study in order to (a) guarantee enough space for unconfined tests, and (b) avoid influences from the edges. All the tests were performed for both investigated concrete ages, namely 28 and 70 days.A 100 mm long post-installed undercut anchor is investigated in the present study. In a first step, a cylindrical hole opening up in a conical undercut is created with a special drill bit. After inserting the anchor consisting of a metal sleeve and a threaded bar ending in a cone, a given installation torque is applied. In this process the anchor adapts to the conical void by which mechanical interlock is achieved. A qualitative sketch of the undercut anchor is shown in The concrete craters left in the slabs after performing the pull-out tests have been digitalized by photogrammetric means in order to be able to comprehensively investigate the aggregate influence including the geometry of the failure surface.In order to investigate the potential aggregate effect on the undercut anchor capacity in tension, three concrete mixes were tested. These three concrete mixes, identified as D1, D2 and D3, were class C25/30 and had the same cement type CEM II 42.5 N. The concrete was delivered by a ready-mix concrete company which optimized also the mix design based on the requirement to alternate the (coarse) aggregate  in the widest range possible while maintaining the rest of the mix design. According to the approval guidelines for mechanical anchors , the con95% (see ).It is commonly known that concrete already approaches it’s full strength at 28 days. In case of the cement used in this experimental campaign, the 28 days compressive strength is around quation  is usedquation : (a) stored with the slab according to the fastenings related guidelines denoted as D (as dry); and (b) stored in lime saturated water until the testing day denoted as M (as moist).E). The moist cure condition was used for cylinders and part of the cubes, and the dry cure condition was used just for the remaining cubes. Fracture tests were performed on moist cured notched beams in a three point bending configuration in order to obtain the total fracture energy  and diameter (d), is Concrete properties were experimentally determined at 28 and 70 days. The elastic modulus was extracted from the loading branch of the cylinder compression tests. The deformation has been measured using three LVDTs  with a base length of 100 mm, placed parallel to the specimen axis, in a 120Compression tests were performed on cubical specimens with an edge length of 150 mm for the two different curing conditions, as shown in Fracture energy is defined as the energy required to create a unit area of crack surface and it is an important property of concrete for investigations concerning fracture and damage propagation. The total fracture energy shown in  is detershown in  is approTensile strength was also obtained experimentally. Brazilian splitting tests were performed on cylindrical slices having a diameter of 150 mm and length of 75 mm. Between the loading plates and the specimen, wooden strips were placed to distribute evenly the load. Such results are available in The results of the pull-out tests of the undercut anchors used are also summarised in The 3D geometrical representation of the crater left from the concrete cone failure is acquired through a photogrammetric tool yielding a 3D point cloud. In order to obtain the crater shape, several pictures have to be taken from different distances and positions. A series of targets are placed around the area to determine the scale and the coordinate system, and to facilitate the recognition of common points among pictures.Once the point cloud is acquired, the cone is divided into slices  versus radius . Points having a radial coordinate value within 15 mm and 30 mm serve as data basis for linear regression . . Nexp) aThe normalisation should isolate the aggregate effect on the pull-out loads from the concrete properties . In case of a perfect prediction model, the normalised pull-out load would approach 1, while a number bigger than 1 indicates a conservative model. The normalised experimental results themselves do not allow drawing a conclusion regarding the existence of an aggregate effect. Such an interpretation can only be based on the comparison between different batches.In this investigation, the relative difference Equation  with αDiFor the mean value analysis just Equation  is consifound in  during aThe fact that in this and most experimental campaigns the number of tests are limited (here to 3 or 5 specimens for each configuration), constrains the certainty of the conclusions if only the mean values are used. Additional insights can be gained by using the information about the scatter in the experiments. Furthermore, it has to be noted that if a function quations , f(x)¯≠fEquation .(8)α(·)=With the same procedure, also the combination of the results based on Equation  can be on) and not the sample version  are used in the standard prediction Equation  beyond what the established model equations can predict. Thus, the current assessment and design framework, which accounts for a variation of the concrete cone capacity as a function of concrete compressive strength only, serves as a safe design method.


We conduct textual analysis of a sample of more than 200,000 papers written on HIV/AIDS during the past three decades. Using the Latent Dirichlet Allocation method, we disentangle studies that address behavioral and social aspects from other studies and measure the trends of different topics as related to HIV/AIDS. We show that there is a regional variation in scientists’ approach to the problem of HIV/AIDS. Our results show that controlling for the economy, proximity to the HIV/AIDS problem correlates with the extent to which scientists look at the behavioral and social aspects of the disease rather than biomedical. Later, scientists uncovered the modes of transmission and developed blood tests for diagnosing HIV3. By the early 1990s, the first antiviral drugs were developed to suppress HIV3. Together, these developments and efforts to educate vulnerable populations about the transmission mechanisms slowed down the progress of the disease during the mid-1990s, at least in the United States. Yet, it was also in the decade of the 1990s that the HIV/AIDS epidemic reached its peak in Sub-Saharan Africa1. Eventually, as a result of global and regional efforts to control the epidemic, both the HIV incidence and AIDS-related death rates have dropped after the year 20004.Since it was first detected in the early 1980s, AIDS has been studied by scientists around the world. Biomedical and epidemiological scientists have made substantial progress in understanding and controlling the disease over time. In the early years, HIV was discovered to be the cause of AIDS5, risk perception6, high-risk behaviors7, willingness to be tested8, social stigma9, and treatment adherence10. The infectious nature of the disease also adds to the social network complexity of the problem and to infection patterns11. Numerous multi-disciplinary studies have been conducted to uncover these socio-behavioral aspects of the disease.The complexity of the HIV/AIDS problem goes beyond biomedical aspects of the disease. Many challenges are behavioral and social, such as public awareness of the disease12. Along the same line of research, preventive behavioral studies have focused on reducing the chances of being infected by HIV and alleviating the adverse outcomes for patients with HIV13. These studies have examined how people can avoid risky behaviors by becoming more knowledgeable, motivated, and capable in the context of avoiding HIV infection7. HIV/AIDS is a disease with strong social stigma attached to it in many societies. Sociologists and anthropologists have contributed to HIV/AIDS research by proposing theories and models to understand and overcome this stigma9. Overall, the behavioral and social sciences (BSS) studies have helped in shaping a better understanding of the disease, recognizing people with the highest risk, designing preventive programs, and maintaining medical treatment for current patients.From the early years, behavioral studies have shed light on the psychological factors corresponding to risky behaviors that can lead to HIV infectionCumulatively, studies conducted by scientists within the fields of biomedical sciences and BSS have shaped our understanding of HIV/AIDS as a global problem. Despite this ostensible shared understanding, the knowledge production process orbiting around the problem of HIV/AIDS has happened incrementally over decades of research and within the context of scientific enterprise in many countries around the world. Yet, little is known about the trends of research and shifts in focus of researchers as related to the problem of HIV/AIDS. In this study, we investigate shifts in the scientific community’s focus on BSS aspects of HIV/AIDS through a textual analysis of more than 200,000 HIV/AIDS publications in the Scopus data set. Our goal is to evaluate global and regional trends and potential variations in research. One finding of our study is that, in the case of HIV/AIDS, the share of BSS research has been increasing over time and, controlling for the economy, geographical proximity to the problem is associated with more BSS studies.We constructed a dataset of all academic papers on the topic of HIV/AIDS as reported in the Scopus data set, totaling 264,102 papers. These papers were published on a variety of subjects related to HIV/AIDS during the period 1985 to 2012. Our criteria for inclusion of the papers in our dataset were existence of the words “HIV” or “AIDS” in the title or abstract of papers. For the analysis, we used the abstract of the papers, year of publication, and location of authors based on their institutional affiliations. Locations of authors were used to assign a paper to one or multiple countries. For example, a paper with authors from three different countries was assigned to those three countries. In the first stage, we excluded papers lacking an abstract, which reduced our dataset to a total of 209,608 papers.We first conducted a set of descriptive analysis at the aggregate level, as reported in Fig. In the next step, we conducted textual analysis, looking at the abstract of HIV/AIDS publications over time. Methodologically, we implemented the Latent Dirichlet Allocation (LDA) on our data set. More information about LDA is provided in the method section; in short, in the LDA method, topics are identified based on the frequency of appearance and co-appearance of different words within all the documents. Our textual analysis of all paper abstracts shows an increasing trend in the share of BSS among HIV/AIDS publications Fig. . In 2012We also tracked the trends of individual BSS topics over time. As examples, Fig. Different countries have shown different level of focus on BSS in their HIV/AIDS research portfolio, as Fig. We observe a regional variation in BSS study trends, as Fig. Regional variation also exists among individual topics. Figure Why does this variation between different regions in terms of their focus on the BSS aspects of HIV/AIDS exist? To study what drives BSS focus in HIV/AIDS research, we perform a panel data regression analysis. The factors we include in our analysis are the HIV/AIDS mortality rate and GDP per capita. We expect HIV/AIDS mortality to be associated with how researchers and science policy makers perceive the problem of HIV/AIDS. We represent the magnitude of the problem by HIV/AIDS mortality rate using the HIV mortality data from the “Global burden of disease study 2013” [Available from http://ghdx.healthdata.org/global-burden-disease-study-2013-gbd-2013-data-downloads]. Our BSS-related question is whether the higher rate of the problem is associated with higher BSS studies.One other potential explanation for research focus is the research capacity or financial resources available for research. Biomedical research is more costly. By having more resources available for research, we expect that affluent countries be more capable of conducting biomedical research. This can affect the relative share of BSS in research portfolio of these countries. In our analysis, we control for GDP per capita as a proxy for countries’ economic prosperity.ρ = 0.58 for relationship between percentage of BSS and HIV/AIDS mortality rate and ρ = −0.56 for relationship between percentage of BSS and GDP per capita). Our analysis also provides evidence that supports a negative correlation between GDP per capita in countries and the ratio of BSS topics in their publications (p < 0.001).Our regression analysis shows there is a positive association between countries’ HIV/AIDS mortality rate and the ratio of BSS topics in their research publications on HIV/AIDS (p < 0.001). The detailed results are reported in the methods section. For the sake of simplicity, Fig. By constructing and analyzing a large data set of more than 200,000 papers, and by implementing a statistical natural language processing method (Latent Dirichlet Allocation), we were able to investigate the trends of studies in HIV/AIDS research in different regions. We found interesting trends in behavioral and social studies of HIV/AIDS. We observe that there is variation among different countries in terms of their focus on HIV/AIDS. The BSS focus has, on average, been the highest in Sub-Saharan Africa. Furthermore, we find an association between HIV/AIDS mortality and the percentage of BSS topics in HIV/AIDS research.If we assume scientists’ responses to a problem are informed by the nature of the problem they observe in their proximity, our findings will have several implications. The scientific community’s focus on the “same” medical disease may vary across different regions and times, and this could speak to the varying nature of a seemingly similar disease in different regions and different time periods. If one assumes that a society’s understanding of a problem materializes in the form of scientific publications, our findings imply that the problem of HIV/AIDS is conceived to be more behavioral and social in places with higher mortality rates. The takeaway from this finding for science policy makers is that in defining priorities of research for a specific problem, the problem should not be looked at from an isolated perspective. To the contrary: it is of the utmost importance to nail down the problem in the context of each country and set priorities based on that perspective.15 implies that scientists’ approach to study a disease can partially reflect societal demand. Under this concept, a higher focus on the BSS aspects of HIV/AIDS in a country implies that those aspects of HIV/AIDS are perceived to be more critical in that region. Such premise seems reasonable: the broadly adverse social, cultural, and economic effects of HIV/AIDS on regions with high prevalence has made it a challenging epidemic to be tackled in those regions16. On the other hand, in low prevalence countries, more focus on biomedical studies may reflect the different nature of the same disease in those regions. In countries with less significant health challenges we often observe higher literacy, better quality of life, and more awareness about health. Consequently, less stress is placed on behavioral and social interventions than biomedical solutions. This premise is in line with medicalization and biomedicalization of health as studied by sociologists of health  that generate the entire set of documents. The output of an LDA implementation over a set of documents includes two pieces: a probability distribution over topics for each of the documents and a probability distribution over all unique words within the data set for each of the topics. The first output helps us know which topics are generating a given document and the second output helps us define each of the topics. LDA has been applied in a wide range of studies, including science and innovation studies24. For more information on the method, see ref. Advancements in the data sciences help us conduct a more precise analysis. We address this methodological issue by using a Bayesian statistical technique known as Latent Dirichlet Allocation (LDA) that helps find the latent topics that generate a set of documents25. In some prior topic modeling applications in the context of science and innovation22, researchers have chosen to use 100 topics for their analysis. Following their lead, we also chose 100 topics to implement LDA in our data set. For topic and term smoothing parameters, 0.1 has typically been chosen as the default value22. However, since we are looking for more finely separated topics and since the topic space of our documents is narrower , we reduced the term smoothing parameter to 0.01. This helps us have more discrete topics21. We used the python LDA 1.0.4 package to perform our analysis [Available at https://pypi.python.org/pypi/lda.].In this paper, we use the abstracts of papers as the set of documents to implement LDA. After removing the common words in English by using a standard stop list, we generated the corpus of unique words that generates all abstracts in our data set. The size of this corpus was 17,783,690 unique words. In our analysis we limited the set of unique words only to the ones that have been repeated in at least 100 abstracts. By applying this rule, the size of our corpus was reduced to 11,192 unique words. To implement LDA, three parameters need to be specified: the number of topics, a topic smoothing coefficient, and a term smoothing coefficient. To have a model with a better fit, using large number of topics is recommended. This, however, poses the problem of confronting topics that are indistinguishable in a meaningful way for human understandingMany of the topics generated by the LDA do not necessarily show a discipline or field of research. In fact, each abstract is a mixture of all the topics with different probabilities. However, we expect that, on average, topics belonging to BSS studies will have higher probabilities assigned to them in BSS publications. Based on this assumption, we anticipate that pairs of topics related to a specific body of knowledge are more likely to have co-occurrence probability  in a publication compared with a pair of topics each related to a different fields of study. By making this assumption, we can look at the co-occurrence network of the topics, as uncovered by LDA, to find fields of research within our publication data set.26 was implemented on this network to find the communities of topics corresponding to fields of research. Three clusters were found. Cluster 1 includes topics related to medical case studies; Cluster 2 includes BSS topics; and cluster 3 includes biomedical topics. The individual topics and these clusters are reported in the SI. The robustness of these clusters against variability of input data was confirmed through sensitivity analysis for random sub-samples of papers. Results are presented in the SI. As another robustness check, we also generated a network with the eleven most probable topics of each paper. Each of these eleven topics, on average, have probabilities higher than 1%. Performing Louvain clustering on this network provided us with very similar clusters.We generate the topic network by considering the five most probable topics of each document. Each of these five have, on average, a probability higher than 5%. Moreover, their average cumulative probability is greater than 70%. In our topic network, each node represents a topic. Topics are connected to each other based on their co-occurrence probability in all papers. The Louvain clusteringFinally, we would like to mention that a benefit of using LDA is to count for multidisciplinary works. Since in this method a topic of a paper is a probability distribution over different potential topics, a paper can be considered as x% BSS and y% biomedical. These percentages are estimated based on the words used in the abstracts.i + 1 to i + 5 on the HIV/AIDS mortality rate of countries in year i controlling for their average GDP per capita during those years. Overall, our analysis includes 84 panels (countries with more than 100 papers on the topic HIV/AIDS during the period 1985-2012) for which we have four observations .To investigate the variation among different countries in terms of their focus on BSS aspects of HIV/AIDS, we performed a panel data regression analysis. As mentioned earlier, we had access to publication data for the period 1985 to 2012. The HIV/AIDS mortality rate estimates are available for the years 1990, 1995, 2000, 2005, 2010, and 2013. In our analysis, we regressed the average share of BSS topics during years 28 in our main model (Model 1). Due to the nature of our problem, we are concerned about heteroscedasticity and autocorrelation in our regression analysis. However, the selected model is robust against heteroskedasticity and autocorrelation. Other potential models such as Huber-White and Newey-West have also been used to confirm our regression results (see the results in SI). Due to the small number of observations per panel, we did not use a fixed effect model in our analysis, as we did not have enough variation. We tested for multicollinearity in our data and did not find an evidence for a potential problem (un-centered VIF: 1).Table 21, we selected Model 1 as our primary model in this paper. Overall, our models offer consistent results showing a positive association between HIV/AIDS mortality rate of countries and their focus on BSS aspects of HIV/AIDS. Our models also provide evidence to support a negative association between GDP per capita of countries and share of BSS in their HIV/AIDS publications.As a robustness check for our model selection, we performed the same analysis in Model 2 with another approach. In Model 2, we used a generalized least square model with heteroskedastic panels and first order autoregression. As Table AppendixDataset


This paper evaluates the influence of two superplasticizers (SP) on the durability properties of concrete made with fine recycled concrete aggregate (FRCA). For this purpose, three families of concrete were tested: concrete without SP, concrete made with a regular superplasticizer and concrete made with a high-performance superplasticizer. Five volumetric replacement ratios of natural sand by FRCA were tested: 0%, 10%, 30%, 50% and 100%. Two natural gravels were used as coarse aggregates. All mixes had the same particle size distribution, cement content and amount of superplasticizer. The w/c ratio was calibrated to obtain similar slump. The results showed that the incorporation of FRCA increased the water absorption by immersion, the water absorption by capillary action, the carbonation depth and the chloride migration coefficient, while the use of superplasticizers highly improved these properties. The incorporation of FRCA jeopardized the SP’s effectiveness. This research demonstrated that, from a durability point of view, the simultaneous incorporation of FRCA and high-performance SP is a viable sustainable solution for structural concrete production. Its use has a great environmental impact due to the large amount of natural aggregates and cement required for manufacture. The production of one ton of Portland cement emits between 0.6 and 1.0 tons of COConcrete structures are demolished at the end of their service life, generating what is known as construction and demolition waste. Using deconstruction techniques , very puMost of the research conducted has been focused on the use of CRCA in the manufacture of new structural concrete ,7,8,9,102 emissions required from crushing rock at particle size below 4 mm.For the reasons given above, many international standards allow replacing natural gravel with CRCA but do not allow replacing natural sand with FRCA for structural concrete production . This siEvangelista and de Brito , after aThe aim of this study was to experimentally determine the influence of regular superplasticizer and high-performance superplasticizer on the durability properties of concrete made with different replacement ratios of natural sand with FRCA and propose maximum FRCA replacement ratios for each of the superplasticizers tested. No target durability performance was set for the mixes with FRCA. CRCA were not used in order to separate the influence of the FRCA on the results from that of the CRCA.The importance of this study is that it demonstrates that the use of admixtures significantly improves the durability properties of concrete made with FRCA, whereby the amount of FRCA can be increased. This has the following environmental advantages: (a) the amount of sand collected from rivers and seashores can be reduced; (b) the landfill or illegal deposits of waste concrete can be minimized; (c) the life cycle of concrete can be increased by having a second use in the manufacture of recycled aggregate concrete.3, 2.28 g/cm3 and 2.56 g/cm3 for dry-oven (ρrd), saturated surface dry (ρssd) and apparent (ρa) particle densities respectively, and average values of 9.9% for water absorption at 24 h. A smaller FRCA particle size results in higher water absorption capacity. Guedes et al. [The SEM images of FRCA particles show a rough surface with mortar adhered to it and numerous pores and micro-cracks  that maks et al.  also fouTo compensate for the greater water absorption and internal friction between particles derived from its greater angularity, concrete made with FRCA requires more total water than conventional concrete for mixing. This increase in the total w/c ratio is not necessarily harmful, since excess water can be absorbed by the FRCA. However, the increase of water deriving from the greater friction between particles generates an increase in the effective w/c ratio . ConcretThe amount of mixing water can be reduced using superplasticizers. The lower w/c ratio improves the compacity of concrete and its mechanical properties ,19. Howenssm) grows linearly when replacement ratio increases, obtaining values ranging between 17.99 × 10−12 m2/s for RC and 24.07 × 10−12 m2/s for 100% FRCA-concrete. The carbonation depth showed a similar trend to chloride penetration. Increases of 40% and 110% were observed for 30% and 100% replacement ratio respectively.Evangelista and de Brito ,12, usinKou and Poon  in a firIn a second study, Kou and Poon  replacedZega and Maio  studied Sim and Park  made a ret al. [Pereira et al. ,19 conduGeng and Sun  evaluateTwo commercially available superplasticizers widely used in Portugal were selected to study the influence of superplasticizers on the durability of concrete made with FRCA: Sikament 400 plus (SP1) and SikaPlast 898 (SP2).3 and 1.07 ± 0.02 kg/dm3 respectively, and their pH (23 ± 2 °C) is 9.0 ± 1.0 and 5.0 ± 1.0, respectively.SP1 is a regular superplasticizer chemically based on organic polymers and admixtures that works by electrostatic repulsion. SP2 is a high-performance superplasticizer chemically based on a combination of modified polycarboxylates in an aqueous solution that works by electrostatic, and basically, by steric repulsions. Their density (23 ± 2 °C) is 1.22 ± 0.02 kg/dmC0: concrete made without SP;C1: concrete made with SP1;C2: concrete made with SP2.Three families of concrete were made:et al. [Five replacement ratios of fine natural aggregate (FNA) with FRCA were tested for each of the three families of concrete (Ci): 0%, 10%, 30%, 50% and 100%. These percentages are consistent with the replacement ratios used in two previous works by Pereira et al. ,19. A toTwo commercial limestone crushed gravels: medium gravel 6/12 mm (CNA1) and coarse gravel 12/20 mm (CNA2), two commercial washed siliceous sands: fine sand 0/2 mm (FNA1) and coarse sand 0/4 mm (FNA2) and one FRCA obtained from crushed concrete blocks were used to manufacture the concrete mixes.max 22 S2 .In order to control the original properties of the concrete, a mix of concrete type X0 (P) CL0.40 D6-1:2007 ) was man6-1:2007 : 1931 kgssd/ρrd value and much higher water absorption.Commercial natural aggregates showed more homogeneous grading curves than FRCA . ContinuThe mixes were designed according to the Faury method. According to NP EN 206-1:2007  the follCriterion-1: Adjust the particle size based on Faury’s method. With the aim of obtaining a curve of maximum compacity and eliminating the effect of particle size on the properties of the concrete, the mass of each of the aggregates was calculated beforehand, according to the Faury method. Fine aggregates were sieved and separated in the following particle sizes: 0.063 mm, 0.125 mm, 0.25 mm, 0.5 mm, 1 mm, 2 mm and 4 mm. The amounts for each of these fractions were incorporated into the mix thus achieving a perfect fit to the Faury curve.FRCA—Mass of FRCA (kg);MFNA—Mass of FNA (kg);Mi—FRCA replacement ratio (%);%FRCA—Oven-dry density FRCA (kg/dm3);δFNA—Oven-dry density FNA (kg/dm3).δFine natural aggregates were replaced by volume for each of the size fractions according to the following equation:Criterion-2: Use a fixed amount of superplasticizer in each of the concrete families. The superplasticizers SP1 and SP2 were added at a fixed proportion of 1% by weight of cement; this value is within the range recommended by the manufacturer of the products. Both admixtures meet the technical specifications of the UNE-EN 934-2:2001 . The admCriterion-3: Adjust the water/cement ratio to obtain a similar slump. The amount of water was adjusted to achieve a similar slump (using the Abrams cone) of 125 ± 15 mm in all mixes tested. The higher FRCA water absorption should be compensated in mixes made with this recycled material. For this purpose the FRCA water absorption over time was studied beforehand, following the method proposed by Leite . In the Criterion-4: No pre-saturated FRCA.et al. [et al. [et al. [Based on the studies of Ferreira et al. , Poon et [et al.  and Tam  [et al. , FRCA weThe evaluation of the fresh concrete properties is fundamental since they have a significant impact on the performance and durability of hardened concrete. Two properties were tested in the fresh state: workability (using the Abram cone) according to NP EN 12350-2:2006  and the The compressive strength according to UNE EN 12390-3:2009  was measThe following durability properties of concrete were studied: water absorption by immersion, water absorption by capillarity, chloride penetration and carbonation resistance.Water absorption by immersion measures the capacity of a fluid to move through the pore structure of concrete and it is mainly related to the open pore structure. This is one of the most important properties in terms of durability. Water absorption by immersion was tested according to standard LNEC E394:1993 .Capillary absorption is a phenomenon by which a fluid moves through the microstructure of concrete induced by the interaction between molecules of the fluid and the surface pressure of the concrete’s capillaries. According to Jurin’s law, capillary movement increases with decreasing capillary diameter. Capillary absorption is related to the diffusion of chloride and carbonation of concrete . This pr2 concentration of 5% ± 0.1%, temperature of 23 ± 3 °C and relative humidity of 50% ± 5%. Each specimen was broken into four parts and immediately sprayed with a phenolphthalein solution. The maximum and mean carbonation depth was measured in each fragment . Three cylindrical specimens with a diameter of 100 mm and a height of 50 mm cut from specimens of 250 mm height and the same diameter were tested. Samples were cured for 14 days in a wet chamber at 20 ± 2 °C and relative humidity of 95% ± 5%. After cutting, the samples were placed in a dry chamber at a temperature of 20 ± 2 °C and relative humidity of 50% ± 5%. The tests were carried out at two ages: 28 and 91 days.The presence of chloride ion in concrete is related to the corrosion of steel. From a content of chlorides, the passivation layer on the surface of steel in concrete is destroyed and the conditions for the onset of corrosion occur. An accelerated chloride penetration test was carried out in accordance with standard LNEC E463:2004  Figure . EvangelFRCA) and the percentage of variation due to the use of superplasticizers SP1 and SP2 with respect to the superplasticizer-free concrete (ΔSP).ef were achieved in concrete made with SP2. The (w/c)ef ratio increased with the use of FRCA for the three concrete families, which is explained by the greater angularity of the recycled particles and the need to use more water to achieve the same slump. Concrete made with SP1 reduced the (w/c)ef ratio with respect to RC0 concrete family between 15.7% and 9.09% for replacement rates of 0% and 100%, respectively, while concrete made with SP2 maintained a reduction in the (w/c)ef ratio of around 25% for every replacement ratio. This shows that SP1 lost effectiveness for increasing percentages of FRCA, while SP2 was impervious to that effect.The use of superplasticizers reduced the effective (w/c) ratio for all replacement ratios of FNA with FRCA. The lowest values of (w/c)et al. [Similar results were obtained by Pereira et al. . These aFRCA) and of the superplasticizers (ΔSP).In all concrete families, density decreased when increasing the use of FRCA, which is justified by the lower density of the FRCA .ef ratio in concrete families C1 and C2, although the differences between C1 and C2 were small.For each replacement ratio, the concrete families made with SP1 and SP2 showed higher density than the superplasticizer-free family (C0). This is justified by the lower (w/c)et al. [Most authors have shown that the density of concrete made without admixtures decreases linearly with the rate of recycled aggregates ,44. Pereet al.  showed scm) for each of the mixes and the values of ΔFRCA and ΔSP defined above.For each of the concrete families , the compressive strength decreased for higher FRCA contents, except for concrete family C0 at 10% replacement ratio, which can be explained by the possible filler effect of the very fine broken particles from FRCA generated during the mixing process that offsets the higher porosity of the recycled aggregates . The comef ratio of the mixes made with SP  with SP . It is sthan RC0 .et al. [Pereira et al.  found a In relative terms the water absorption by immersion was similarly affected by the use of FRCA in the three families of concrete, since the slopes of the linear trends were very similar ; as can Using the results of the 15mixes, a direct relationship between water absorption by immersion and effective w/c ratio can be observed . FurtherAccording to the literature review, manufacturing of concrete made with FRCA without admixtures or conventional superplasticizers significantly increased the water absorption by immersion parameter ,22. So, The evolution over time of the capillarity water absorption for each of the concrete families and replacement ratio of FRCA is shown in de Brito , the watIt is clear that the capillary water absorption increased with the incorporation of FRCA. This property is basically determined by the porosity of concrete, which increases with the percentage of FRCA. Two reasons are given for this greater porosity in concrete made with FRCA: the FRCA’s greater porosity and the higher effective (w/c) ratio required to achieve the target slump value .ef ratio 7.8% relative to the reference mix (RC0).A higher increase was noticed for the 100% replacement ratio, which is explained by the exponential growth of the connecting capillaries for this replacement ratio, to which Evangelista and de Brito  refer. ef ratio by 16.3% relative to the reference mix (RC1). The family made with SP2 (C2) showed the greatest difference in the 72 h test, 15%–95% for 50% and 100% of replacement ratios: The mix made with 100% FRCA  increases the (w/c)ef ratio by 7.9% relative to the reference mix (RC2). A linear increase of the capillary with FRCA can be adjusted very well  increased from 31% to 58% for 50% and 100% of replacement ratios. The mix made with 100% FRCA  increases the (w/c)ery well .Concrete families made with SP showed the lowest values of sorptivity . For eacef ratio 15.7% and 25.5% lower than RC0  needed a (w/c)than RC0 .i.e., 43% and 54% for C1.100 and C2.100. In this case, the mixes C1,100 and C2,100 needed a (w/c)ef ratio 9.1% and 25.5% lower than RC0. Mixes incorporating admixtures are more sensitive to the incorporation of FRCA due to the distribution changes caused by the SP, referred to by Mehta and Monteiro [The capillary water absorption values were slightly lower when 100% FRCA was used, Monteiro , which eA liner correlation between the capillary water absorption and the water absorption by immersion can be observed . The relvs. replacement ratio is shown in The carbonation depth for each of the concrete families In relative terms, the superplasticizers’ efficiency is compromised by the incorporation of FRCA, especially at early ages. This can be justified by the higher porosity of FRCA, which results in a greater contact surface between the aggregate particles and superplasticizers. The content used in the reference mixes RC1 and RC2 is insufficient to maintain the effectiveness of the superplasticizers. This is more evident in the SP2 family, as shown by the higher slope of the linear trend ; this isThe carbonation depth is proportional to the effective w/c ratio  and inveThe chloride penetration test results at 91 days are shown in Maximum decreases of 38% and 46% were obtained for the concrete families made with SP1 and SP2 respectively, which was related to their lower effective w/c ratio. FRCA and ΔSP. Concrete made with 100% FRCA and SP2 showed a chloride diffusion coefficient value of 10.60 × 10−12 m2/s, whereas the reference concrete RC0 showed a higher value of 12.57 × 10−12 m2/s. As in the carbonation depth, the loss of porosity achieved with the use of SP2 was greater than the increase resulting from the use of FRCA and the higher effective w/c ratio necessary in the mix to obtain similar slump.The chloride diffusion coefficient was proportional to the effective w/c ratio  and inveThis paper demonstrates the feasibility of using FRCA in structural concrete production from a durability point of view. Three families of concrete were tested: concrete without SP, concrete made with a regular superplasticizer and concrete made with a high-performance superplasticizer. In each family, five volumetric replacement ratios of natural sand by FRCA were tested: 0%, 10%, 30%, 50% and 100%. The following conclusions can be drawn:The effective water/cement ratio increased up to 16%. The effect of FRCA was more pronounced in the family of concrete made with the regular superplasticizer;The water absorption by immersion increased linearly up to 52%. This property was similarly affected by the percentage of FRCA in the three families of concrete;The water absorption by capillary action in the 72 h test increased up to 45%. The effect of FRCA was more pronounced in the family of concrete made with the high-performance superplasticizer;The carbonation depth at 91 days increased up to 333%. The effect of FRCA was more pronounced in the family of concrete made with the high-performance superplasticizer;The chloride migration coefficient increased up to 33%. The effect of FRCA was more pronounced in the family of concrete made with the high-performance superplasticizer.1. The incorporation of FRCA up to 100% had the following consequences on the concrete’s durability properties:The effective water/cement ratio decreased up to 16%. This percentage of variation decreased with the incorporation of FRCA;The water absorption by immersion decreased up to 28%. This percentage of variation was slightly reduced by the incorporation of FRCA;The water absorption by capillary in the 72 h test decreased up to 48%. This percentage of variation was reduced by the incorporation of FRCA;The carbonation depth at 91 days decreased up to 59%. This percentage of variation was strongly reduced by the incorporation of FRCA;The chloride migration coefficient decreased up to 38%. This percentage of variation was reduced by the incorporation of FRCA;The regular SP’s effectiveness was significantly jeopardized by the incorporation of FRCA.2. The addition of a regular superplasticizer had the following consequences on the concrete’s durability properties:The effective water/cement ratio decreased up to 25%. The effect of the high-performance superplasticizer was not significantly affected by the incorporation of FRCA;The water absorption by immersion decreases up to 43%. This percentage of variation was not significantly affected by the incorporation of FRCA;The water absorption by capillary in the 72 h test decreased up to 66%. This percentage of variation was reduced by the incorporation of FRCA;The carbonation depth at 91 days decreased up to 80%. This percentage of variation was reduced by the incorporation of FRCA;The chloride migration coefficient decreased up to 46%. This percentage of variation was reduced by the incorporation of FRCA;i.e., the effect rises as the admixture’s quality increases.The high-performance SP’s effectiveness was much more jeopardized than regular SP by the incorporation of FRCA, 3. The addition of a high-performance superplasticizer had the following consequences on the concrete’s durability properties:4. In view of the results, the maximum FRCA replacement ratios that allow maintaining the RC’s performance for each of the durability properties are shown in 5. In conclusion, the simultaneous incorporation of FRCA and high-performance SP is a viable sustainable solution for structural concrete.


AbstractAlburnussava, new species, is described from the Kolpa River. The Kolpa is a tributary of the Sava, a major tributary of the Danube River, in the Black Sea basin. Alburnussava is distinguished from its congeners in the Danube drainage, A.mento and A.sarmaticus, by having 23−27, usually 24−26, gill rakers; the ventral keel usually completely covered by scales ; 15−16, mode 15, branched pectoral-fin rays; the length of the gill raker at the junction of the arch limbs 65−70% of the length of the opposite outer gill filament; and a relatively long lower jaw . Alburnussava is a large-sized potamadromous shemaya known to occur in the entire Sava drainage. The taxonomic status of A.mento and A.sarmaticus is confirmed. Alburnusdanubicus is discussed and as there are no new arguments, it is kept as a valid species. New details on the distribution of shemayas in the Danube drainage are presented. It is also commonly applied to a number of nominal species and subspecies in the Caspian and the Black seas since Chalcalburnus Berg, 1932 and discriminated from Alburnus by a usually scaled  ventral keel between the pelvic fins and the anus  and an increased number of lateral-line scales, usually over 55   , 1933. Rp to 63)  supporteic group .A.danubicus Antipa, 1909 (anadromous), A.mento  (landlocked), A.sarmaticus Freyhof & Kottelat, 2007b (anadromous and potamadromous), A.mentoides Kessler, 1859 (resident), A.leobergi Freyhof & Kottelat, 2007b , A.derjugini Berg, 1923 PageBreak(resident), A.istanbulensis Battalgil, 1941 (resident), A.schischkovi  (anadromous), and A.mandrensis  . Shemayas distributed outside of the Black Sea basin include A.volviticus Freyhof & Kottelat, 2007a and A.vistonicus Freyhof & Kottelat, 2007a, both from lakes in eastern Greece, Aegean Sea basin (landlocked); two species from lakes in the Sea of Marmara basin in Turkey, A.carinatus Battalgil, 1941 (landlocked) and A.nicaeensis Battalgil, 1941 ; two species in the Aegean Sea basin in western Turkey, A.attalus Özuluğ & Freyhof, 2007 (resident) and A.battalgilae Özuluğ & Freyhof, 2007 (resident), and A.chalcoides from the Caspian Sea basin (anadromous).Earlier authors e.g.,  emphasizA.derjugini and A.istanbulensis . It is still to be clarified if there is (was) a single species in the Çoruh as supposed by However, this taxonomic scheme still does not answer all remaining questions for the group. For example, it is not clear which species occurs between the ranges of A.chalcoides and the Black Sea A.mento.Current knowledge of the morphology of the Black Sea shemayas is inadequate as the keys offered to distinguish them  are ofteA.sarmaticus, but differing in the number of gill rakers . A morphological analysis of additional material from the Kolpa is important in resolving the taxonomy of the Danubian shemayas. Herein, we examine morphological variation of shemayas and provide a critical review of their historical distributions in the Danube.As to the Danube drainage, as indicated above, PageBreakmost measurements follow The triangular-shaped symphisis of the lower jaws is referred as the chin. The ventral keel is defined as the distance between the base of the anus and the level of the posterior ends of the pelvic-fin bases. The dorsal-fin insertion is the posterior-most point where the last dorsal-fin ray connects with the body. All measurements were made point-to-point with a dial caliper and recorded to the nearest of 0.1 mm. Methods for counting rays, lateral-line scales and scales along the scaleless part of the ventral keel, and for SL, standard length; HL, lateral head length; HDBI, Croatian Biological Research Society; MNCN, Museo Nacional de Ciencias Naturales ; NMW, Naturhistorisches Museum Wien ; vs., versus; ZM NASU, Zoological Museum of National Academy of Sciences of Ukraine.Abbreviations: CA), Multidimensional Scaling (MDS), Principal Component Analysis (PCA), Discriminant Function Analysis (DFA), and a Kruskal-Wallis test which is helpful for comparison of three and more groups with a presumably non-parametric distribution of variables, were performed using STATISTICA 6.0 and PRIMER v6.1.9 to identify the most important characters that contribute to the differentiation of samples and visualise the degree of morphological separation among the new species, A.mento, A.sarmaticus, and A.leobergi.Cluster Analysis , Slovenia, 3 Oct 2013, coll. B. Levai.PageBreakMNCN 291346-53, 8, 105−151.5 mm SL, same data as holotype; HDBI 255, 218 mm SL, Kupa [Kolpa] River at Ozalj (45.62°N 15.47°E), Sept 2011, Croatia, coll. D. Jelić; HDBI 1224, 3, 62.9−79.8 mm SL, same data as HDBI 255.Alburnussava sp. n. is distinguished from all other species of Alburnus in the Danube drainage by having 23−27, usually 24−26, gill rakers; the ventral keel usually completely scaled ; 15−16 (mode = 15) branched pectoral-fin rays; the length of gill raker 65−70% of the length of the opposite outer gill filament; and a relatively long lower jaw .Alburnussava sp. n. can be seen in Figure SL. As the examined samples are rather small in number of specimens and contain individuals with a wide range in SL, Table SL and considerably exceeded the range in body depth of smaller specimens (20−23% SL). The same is found for the depth of the caudal peduncle, 10% SL  vs. 9% , respectively. However, head length and eye diameter are clearly negatively allometric. The head length mean in the small-size group (63−80 mm SL) is 25.8% SL vs. 22.7% SL in specimens 174 and 218 mm SL; in smaller specimens the head length considerably exceeds the body depth while in the largest specimen it is much smaller than the latter. The eye diameter mean is 7.5% SL (29% HL) in the small-sized group (63−80 mm) vs. 5.1% SL (22.5% HL) in 218 mm long specimen.The general appearance of The mouth is upturned and the mouth cleft is straight. The tip of the mouth is about at a level with the upper margin of the pupil. The lower jaw is long, its length 112−130% interorbital width. The chin is variably developed  or scaleless (exposed) for 1−2 scales only  length of gill raker 65% and 70% 63−64, mode 63; lateral-line scales to the posterior margin of hypurals 57−62, mode 60. Total vertebrae 44−45, 23−24 abdominal and 20−21 caudal , which includes A.mento, A.sarmaticus, and A.leobergi, as defined by Alburnussava sp. n. in examined materials is 218 mm SL , which is about the body size of mature adults of migratory shemayas in the Black Sea basin, including the Danube − ca. 200−300 mm  (The new species belong to the ntoides) .Alburnussava sp. n. does not demonstrate a distinct difference in most morphometric characters from all or either of the other Danubian species and A.leobergi ; a longer lower jaw, 37−40% HL or 112−130% interorbital width ; and a longer cranial roof, 14−16% SL (vs. 12−15% SL) , should be examined to clarify the problem.A.mento, A.sava sp. n. with 15½ or 16½ branched anal-fin rays do not differ from the former species possessing 13−18½ branched anal-fin rays . Third, 15−16, with a mode of 15, branched pectoral-fin rays that is the lowest count among the examined samples (Table A.mento).When compared to ys Table . Three mAlburnussava sp. n. and A.mento are distinguished in a much clearer way by the length of the scaleless portion of the ventral keel and the relative length of a gill raker. As shown by Alburnus . In A.sava, the ventral keel is completely scaled or there is a very short scaleless portion of the keel which is only 1 or 2 scales long, maximum 15% of the keel length . In contrast, in A.mento  and 23−27, usually 24−26, gill rakers (vs. 25−35) and from A.schischkovi by having 57−62 lateral-line scales to posterior margin of hypurals (vs. 63−67). Alburnussava sp. n. is also distinguished from A.istanbulensis by having the length of the gill raker 65−70% of the opposite outer gill filament (vs. 30−55%) and 23−27, usually 24−26, gill rakers .A.mento.Note on syntypes of  A comment should be given clarifying the status of the specimens labelled as syntypes of A.mento in NMW. The original description (NMW. The set of Acquisition Sheets (bound in a number of books at present) is the most reliable source of primary original information which accompanied the samples at the moment of their accession in the collection.cription : 225−226NMW (16261 and 16441), and one was sent to the Paris Museum. The two NMW specimens have a standard length  which corresponds to a total length equaling “Spanne” [Handspanne] (the distance between the end of the little finger and the end of the thumb that is about 18−22 cm), mentioned in the original description.1. Specimens collected by Heckel in September 1824 in Lake Traun . Heckel’s specimens collected in September 1824 are registered under the acquisition number 1824.II.10: «Traun, … Heckels Reise durch Oberösterreich… Nr. 80». Two specimens are still in PageBreaktion 1830.II contains 7 entries in total  and reads «Bavaria. November 1829. Von Herrn Leopold Fitzinger durch Kauf». This acquisition is made by Jos. Natterer and 1830.II.3 refers to Leuciscusmacroramphus Agassiz with the name AspiusHeckelii Fitz. handwritten later by Heckel. “4-6” Individual: (?) 4 were sent somewhere on exchange [in Tauch]. The labels for NMW 50440, 55650, and 55652 (with the acquisition number 1830.II.3) reading «Durch Agassiz aus München» appeared later, at Steindachner’s time, and are most probably based on information from the Heckel’s description of Aspiusmento Agassiz as a synonym of Aspiusheckelii Fitzinger  from the Danube near Vienna. This specimen was registered under the acquisition number 1836.I.19: «Danube at Vienna. November 1835».NMW 55629, does not match the original description. It contains four specimens registered under the acquisition numbers 1835.VI.1 (2 specimens) and 1835.VI.1a (2 specimens) which say that the specimens were received by exchange from [Dr] Schreibers in 1832 and 1834, respectively, from near Stift Kremsmünster . However, the source of the acquisition may mean any water body in the surroundings of Kremsmünster where an astronomical and geophysical observatory , the first in the world, was located which included a natural history museum with botanical and zoological collections (still existing as a weather station). However, the label (from Steindachner’s time) says «Traunsee» . As Kremsmünster was not mentioned in the original description by NMW 55629 specimens do not belong to the type series and the designation of the 150 mm long (SL) specimen from this sample as lectotype by The accession information for one more sample labeled as syntypes, PCA and MDS analyses applied to 16 meristic variables outlined three clusters  vs. less than 0.1025 for all other variables. A Kruskal-Wallis test revealed four (including the two mentioned above) characters different on a statistically significant (0.01%) level: the number of scales above the lateral line =18.0313487 p=0.0004); the number of branched pectoral-fin rays =19.2995932 p=0.0002); the number of gill rakers =34.8078209 p=0.0000001), and scales along the scaleless portion of the ventral keel =29.1477545 p=0.00002).Based on the DFA, a Kruskal-Wallis test was applied and 10 characters were excluded as not significantly different (p>0.01%) between the samples. Among other 37 characters, a PCA revealed 15 variables whose contribution to either of Factors 1 and 2 or both was the highest (over an arbitrary fixed threshold of 0.01 based on the number of the used variables and their variance); these characters are given in bold in Tables DFA   =16.661, p<0.0000, which indicate almost perfect discrimination. Alburnussava sp. n. is the most distant from A.mento ; A.sarmaticus+A.leobergi is much closer (54.11) but still significantly distant. The most removed, by the characters considered, are A.mento and A.sarmaticus+A.leobergi (107.02). Partial Lambdas demonstrate the unique contribution of the respective variable to the discriminatory power of the whole model, and the most significant  for discrimination of the samples under consideration were number of scales along scaleless portion of ventral keel (0.481705), cranium width between margins of sphenotics (% cranium roof length) (0.545483), cranium width between margins of pterotics (% cranium roof length) (0.803940), number of gill rakers (0.810206), number of branched pectoral-fin rays (0.830673), number of scales above lateral line (0.845142), length of lower jaw  (0.878840), and body depth at dorsal-fin origin (% SL) (0.886565).As a further step, the samples of ers Figs , 9 and ave) Fig. ; predictPageBreakShemayas are reported in the Danube from the upper reaches down to the delta Fig. .A.mento, was known to occur (or still occurs) in lakes in Germany in systems of the Isar (Starnberger See [Würmsee]) and the Inn , in Austria in the Traun River system  and the Drava [Drau] system (Wörthersee [Vrba]), and in Slovenia in the upper Sava   from different lakes likely have a polyphyletic origin as found for lake Coregonus  and its taxonomic implication is beyond the scope of this paper.In the upper Danube, a shemaya, commonly identified as Jezero]) . Most ofrt River . Freyhofake Bled  in the vus e.g., . HoweverNMW 55630, a syntype); this specimen may represent the anadromous or potamadromous Danubian species rather than the landlocked A.mento (NMW specimen labeled as A.mento from the Danube at Lobau near Vienna (NMW 54831) but it is a Leuciscusaspius. Shemayas were also known to be found occasionally in the Hungarian section of the Danube downstream of the Austrian section  was known from near Vienna  the entire Sava River system and, probably, the Danube upstream from the Iron Gates being geographically separated from the anadromous shemaya of the lower Danube (A.danubicus and A.sarmaticus).Formally, the name PageBreakRight below the Iron Gates (a 134-m-long gorge on the main stream of the Danube dammed in 1964 and 1977), shemaya were known in Romania  and the So, historically, shemaya in the Danube were distributed in almost the entire drainage. The length of the Danube and its major tributaries makes it reasonable to suppose that the drainage was historically populated by an anadromous shemaya (entering the river for spawning from the sea) and a resident potamadromous shemaya. At present, it is difficult to assume how far the anadromous form(s) or species used to migrate upstream in the Danube. All anadromous shemayas in the Black Sea and the Sea of Azov basins spend most of the year foraging in coastal sea areas, estuaries, and limans with salinity up to 10−12‰ and wintering in deep places of lower reaches of rivers. Before damming of rivers, they migrated upstream to reach spawning grounds at a distance from tens to hundreds of kilometers depending on the size of the river and availability of grounds suitable for spawning (riffles with gravel bottom and rapid flow). Shemaya were known to migrate up to Pervomaysk in the South Bug, the Dnieper River Rapids (upstream of the town of Zaporizhia) in the Dnieper, and the Oskol River in the Siversky Donets system of the Don River . It lookA.danubicus, A.sarmaticus, and A.sava sp. n. This question can only be solved when specimens from the mainstream Sava and its tributaries in Croatia, Bosnia and Herzegovina, and Serbia are studied as well as specimens from the Danube below its confluence with the Sava. This task cannot be easily implemented because of the rarity of the fish; though, in some tributaries of the Sava in Bosnia and Herzegovina it is still often recorded (No materials were available for us to clarify a border between the ranges of recorded .AlburnussarmaticusZM NASU 4145, 5, 170.7−204.5 mm SL, Ukraine, South Bug River, 2 km downstream of Yuzhno-Ukrainsk, 7−11.05.1989; ZM NASU 2529, 6, 157.5−209 mm SL, Ukraine, Danube, 1922; NMW55507, 3, 178−208.8 mm SL, Lower Danube, Galatz, 1910; NMW55509, 5, 169.4−202.8 mm SL, Lower Danube, Cernavodă, 1910; NMW 55654,1, 175.3 mm SL, Delta of Danube, Vilkovo, 08.1924.PageBreakAlburnusleobergiNMW 6, 154.8−193.1 mm SL, Yasenskiy Bay, Sea of Azov.AlburnusmentoNMW 16261, 1, syntype, 139.8 mm SL, Traunsee; NMW 16441, 1, syntype, 134 mm SL, Traunsee; 50440, 1, syntype, 220.4 mm SL, Bavaria; NMW 50441, 1, 89.2 mm SL, Wörther-See; NMW 55628, 7, 123.8−147.4 mm SL, Gilgen, Wolfgangsee; NMW 55629, 2, 129.2−144 mm SL, Kremsmünster; NMW 55634, 17, 103.3−156.4 mm SL, Traunsee; NMW 55636, 9, 97.1−122.7 mm SL, Attersee; NMW 55642, 4, 148−162.9 mm SL, Gilgen, Wolfgangsee; NMW 55646, 1, 104.4 mm SL, Wörther See; NMW 55648, 2, 112,2−112.9 mm SL, Attersee; NMW 56652, 1, syntype, 210.9 mm SL, Bavaria; NMW 79592, 5, 134.3−143.3 mm SL, Mondsee; NMW 79593, 1, 214.3 mm SL, Mondsee; NMW 80138, 1, 221.2 mm SL, Mondsee; NMW 80622, 1, 191.2 mm SL, Mondsee; NMW 80623, 7, 170.4-220.5 mm SL, Mondsee.


On day 14 from initiation of treatment, intra-tumor MET levels were measured and found to highly correlate with tumor volume, both in the pancreatic cancer PDOX  and melanoma PDOX . Tumors with low concentration of MET were smaller. The present results demonstrates that patient tumors are highly dependent on MET for growth and that rMETase effectively lowers tumor MET.An excessive requirement for methionine (MET) for growth, termed MET dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by MET restriction such as with recombinant methioninase (rMETase). In the present study, we utilized patient-derived orthotopic xenograft (PDOX) nude mouse models with pancreatic cancer or melanoma to determine the relationship between intra-tumor MET level and tumor size. After the tumors grew to 100 mm This phenomena is termed MET dependence . MET res in vivo .11C] MET PET imaging, where high uptake of [11C] MET results in a very strong and selective tumor signal compared with normal tissue background. [11C] MET is superior to [18C] fluorodeoxyglucose (FDG)- PET for PET imaging, suggesting MET dependence is more tumor-specific than glucose dependence ) [EC 4.4.1.11] from ego, CA) , 23. rMEego, CA) , 25.3) = length (mm) × width (mm) × width (mm) × ½ [PDOX mouse models were randomized into two groups: untreated control; rMETase . Tumor length and width were measured at post-treatment. Tumor volume was calculated with the following formula: Tumor volume (mm(mm) × ½ .Each tumor was sonicated for 30 seconds on ice and centrifuged at 12,000 rpm for 10 minutes. Supernatants were collected and protein levels were measured using the Coomassie Protein Assay Kit . Protein levels were calculated from a standard curve obtained with a protein standard, bovine serum albumin (BSA). MET levels were determined with the HPLC procedure described previously. MET levels were calculated per mg tumor protein .P ≤ 0.05 was considered statistically significant.JMP version 11.0 was used for analysis of variance (ANOVA). A probability value of Currently melanoma –59 and p