Leucine-rich repeat kinase 2 (LRRK2) mutations represent the most common genetic cause of sporadic and familial Parkinson’s disease (PD). Especially, LRRK2 G2019S missense mutation has been identified as the most prevalent genetic cause in the late-onset PD. Advanced glycation end products (AGEs) are produced in high amounts in diabetes and diverse aging-related disorders, such as cardiovascular disease, renal disease, and neurological disease. AGEs trigger intracellular signaling pathway associated with oxidative stress and inflammation as well as cell death. RAGE, receptor of AGEs, is activated by interaction with AGEs and mediates AGE-induced cytotoxicity. Whether AGE and RAGE are involved in the pathogenesis of mutant LRRK2 is unknown.Using cell lines transfected with mutant LRRK2 as well as primary neuronal cultures derived from LRRK2 wild-type (WT) and G2019S transgenic mice, we compared the impact of AGE treatment on the survival of control and mutant cells by immunostaining. We also examined the levels of RAGE proteins in the brains of transgenic mice and PD patients by western blots.We show that LRRK2 G2019S mutant-expressing neurons were more sensitive to AGE-induced cell death compared to controls. Furthermore, we found that the levels of RAGE proteins were upregulated in LRRK2 G2019S mutant cells.These data suggest that enhanced AGE-RAGE interaction contributes to LRRK2 G2019S mutation-mediated progressive neuronal loss in PD. Parkinson’s disease (PD) is the second most common neurodegenerative disease with progressive loss of pigmented dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) . AlthougAdvanced glycation end products (AGEs), raised from the reaction of sugars with certain amino acids or fats, are formed in high amounts in diabetes and also in the physiological organism during aging –9. They Based on these earlier studies, we hypothesized that AGE-RAGE interaction might contribute to progressive neuronal cell death in G2019S LRRK2 mutation-expressing cells. In this study, we used LRRK2 G2019S overexpressing mouse model for in vitro neuronal culture experiments and demonstrated that LRRK2 G2019S mutant-expressing neurons were more sensitive to AGE-induced toxicity compared to the controls. In addition, RAGE levels were upregulated in LRRK G2019S mutant cells, suggesting enhanced AGE-RAGE activation is involved in LRRK2 G2019S mutation-related progressive neuronal loss.HEK293 cells were culture in Dulbecco’s modified Eagle’s medium  containing a high glucose concentration supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. For the transfection, Fugene HD (Roche) was used. Primary neurons from cortex and striatum were prepared from newborn pups  . BrieflyTo prepare AGE, BSA (50 mg/mL) was incubated with 0.5 M glucose in 0.2 M sodium phosphate buffer pH 7.4 for 10 weeks at 37 °C. The control sample of albumin was also incubated under same conditions but without glucose. All incubations were performed under sterile environments.To determine the number of viable HEK293 cells, trypan blue dye exclusion assay was performed. It is based on the principle that live cells possess intact cell membrane that excludes dye whereas dead cells do not. A 1:1 dilution of HEK293 cell suspension was mixed with 0.4% Trypan Blue solution (Bio-Rad) at room temperature for 1-2 min. Viable cells showed clear cytoplasm, whereas nonviable cells were blue. Cell mixtures were injected into the hemocytometer chamber, and counted under the microscope. To assess the survival of primary cultured neurons, we seeded the same number of neurons in each culture and randomly captured the images for about 100 MAP2-stained neurons in vehicle and AGR-treated groups. We counted the number of survived neurons based on the appearance of normal dendritic morphology, mainly the continuous and elaborated dendritic trees.The cDNA fragments encoding full-length human G2019S LRRK2 mutant was inserted into a tetracycline operator-regulated gene expression vector, pPrP-tetP. To facilitate protein identification, C-termini of human G2019S LRRK2 protein were tagged by hemagglutinin (HA) epitope. The F1 transgenic mice were crossed with CaMKII-tTA mice to achieve high expression of LRRK2 G2019S in the forebrain . The micFor immunocytochemistry, cultured neurons were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 in PBS. After blocking nonspecific staining using 10% goat serum (Sigma-Aldrich) for 1 h, cells were incubated with primary antibodies overnight. For immunohistochemistry, mice were perfused via cardiac infusion with 4% PFA in cold PBS. To prepare frozen sections, brain was removed and submerged in 30% sucrose for 24 h and then sectioned at 40 μm thickness using cryostat (Leica CM1950). Primary antibodies used included MAP2 (SantaCruz), GFAP (Sigma-Aldrich), and NeuN (Millipore). To detect RAGE protein, primary antibodies from SantaCruz and R&D were used. Anti-LRRK2 polyclonal antibodies (4EC9E and 4C84E) were kindly provided from Dr. Jean-Marc Taymans. Alexa 488 or 568-conjugated secondary antibody was incubated to visualize the staining. Fluorescent images were captured by a Zeiss confocal microscope (LSM 510 META).2, 1 mM NaHCO3, and 0.5 mM CaCl2) containing protease and phosphatase inhibitor cocktail (Roche). To obtain crude membrane fraction, homogenized brain samples were centrifuged at 1000 × g for 10 min to remove nuclear fraction and then supernatants were centrifuged at 20,000 × g for 20 min. The pellet fraction was dissolved in RIPA buffer by sonication and concentrations were measured by BCA assay. Proteins were analyzed by 4-12% NuPage BisTris-polyacrylamide gel electrophoresis (Invitrogen) in MOPS running buffer (Invitrogene) and transferred to polyvinylidene difluoride (PVDF) membranes. The signals were visualized by enhanced chemiluminescence development (Pierce) and quantified using Scion Image System .Mouse brain tissue was homogenized with 10 volumes of sucrose buffer . Statistical significances were obtained by comparing means of different case using t test or ANOVA followed by Tukey’s honestly significant difference test. Error bars indicate SD. To test the toxic effects of AGEs generated for the cell viability assay, we first treated HEK293 cells with increasing dose of AGEs Fig . After tNext, we cultured the striatal neurons (DIV 15) from the striatum of P0 G2019S LRRK2 transgenic (Tg) and wild-type (WT) LRRK2 Tg pups to investigate whether these neurons exhibit different vulnerability to AGEs at 0.1 and 0.15 μg/μl after 24 h treatment Fig. . We founWe then investigated whether AGE-induced neuronal cell death in LRRK2 G2019S neurons is mediated by any specific intracellular signaling pathways. Receptor of AGE (RAGE) is reported to be expressed in aged brains. Therefore, we hypothesized that neuronal cell death triggered by AGE is mediated by RAGE. To test the role of RAGE, we neutralized RAGE function using anti-RAGE antiserum in LRRK2 G2019S neurons Fig. . As a reTo test whether RAGE protein levels are regulated by G2019S LRRK2 expression, HEK293 cells were transfected with doxycycline (DOX)-activated G2019S LRRK2 expression constructs Fig. . We founFinally, we examined the levels of RAGE proteins in the human brain samples from sporadic PD patients and age-matched control subjects. We tested total protein lysates extracted from striatal tissues. As expected, significantly increased RAGE expression in the striatum from sporadic PD patients was detected by the western blotting Fig. . This upDiverse mutations in LRRK2 underlie the most common genetic cause of familial and sporadic PD. Since the identification of PD-associated mutations in LRRK2, many studies have attempted to illustrate the potential pathogenic mechanisms of the neuronal cell death caused by mutant LRRK2, especially the G2019S mutation. Here, we demonstrated that AGE-induced toxicity in the striatal neurons was enhanced in the presence of G2019S LRRK2. Furthermore, we found that the level of RAGE, the critical mediator of AGE-induced cell death, was upregulated in the G2019S LRRK2-expressing neurons. This finding was confirmed in diverse experimental systems, including G2019S LRRK2-expressing cell lines, primary neurons, and mouse brain extracts, as well as postmortem PD striatal tissues. These results reveal a previously unknown pathogenic mechanism of LRRK2 G2019S mutation in AGE/RAGE-mediated cytotoxicity.We detected elevated protein levels of RAGE but not mRNA levels (data not shown) in G2019S LRRK2, indicating the possibility that the upregulation of RAGE mRNA translation by G201S LRRK2, resulting from the pathologically enhanced kinase activity of G2019S mutation. Also, in support that increased RAGE protein levels are not the results from artificially over-expressed G2019S LRRK2, we checked the AGE-induced cell toxicity and RAGE levels in the wild-type LRRK2 overexpressing neurons. We found significant changes of RAGE levels between wild-type LRRK2 and G2019S LRRK2 overexpressing neurons, indicating that this event is not resulted from artificial transgene expression.AGEs, the senescent protein derivatives are formed at accelerated rate under normal aging process, which elicit reactive oxygen species generation and inflammation, and subsequently alters diverse gene expressions, apoptotic neuronal cell death –22. It hIn summary, we demonstrate that AGE-RAGE cascades are involved in G2019S LRRK2-mediated pathogenesis in PD. Our study suggests that targeted pharmacological interventions using inhibitors or antagonist, such as RAGE neutralizing antiserum, or inhibitors against AGE-RAGE intracellular signaling may serve as promising therapeutic strategies to slow down the progression of PD, especially, PD patients having G2019 LRRK2 mutation.


Multiple targeted therapy for advanced clear-cell renal cell carcinoma (RCC) has substantially improved patient outcome, but complete remission is uncommon and many tumors eventually develop resistance. Mechanistic, preclinical, and early clinical data highlight c-Met / hepatocyte growth factor receptor as a promising target for RCC therapeutic agents.MET gene copy gains and MET gene mutation in a large, hospital-based series of renal cell carcinomas with long-term follow-up information.We have examined MET expression, frequency of MET copy number gains were associated with an aggressive phenotype and an unfavorable patient outcome. Elevated protein levels in absence of gene amplification were not attributed to mutations, based on results of targeted next-generation sequencing.Out of a total of 572 clear-cell RCC, only 17% were negative for MET expression whereas 32% showed high protein levels. High MET expression and MET copy number increase is evident in a substantial percentage of patients with high-grade carcinomas and metastatic disease. Diagnostic assessment of MET expression and amplification may be of predictive value to guide targeted therapy against MET signaling in patients with clear-cell RCC.Our data reveal that clear-cell RCC with MET upregulation show an aggressive behavior and  Treatment of metastatic renal cell carcinoma (mRCC) has dramatically changed over the last decade and multiple targeted therapies have replaced IL-2 and IFN-α immunotherapy as the primary treatment option. These new agents, such as Sorafenib, Sunitinib or Temsirolimus, mainly target two pathways, vascular endothelial growth factor (VEGF) signaling and the mammalian target of rapamycin (mTOR) , 4, 5, 6MET encodes for a receptor tyrosine kinase (RTK) called c-Met or hepatocyte growth factor receptor (HGFR). Cells of epithelial origin widely present HGFR on their surface and overexpression of HGFR is common in carcinomas [rcinomas . The ligrcinomas .MET activation include mutation and amplification. Activating germline MET mutations have been observed in patients with hereditary papillary RCCs (papRCC) and in 13% of sporadic papillary RCC [MET have been reported in head and neck squamous cell carcinomas [MET amplifications have been reported in colorectal, gastric, esophageal, lung and clear-cell ovarian cancer [Mechanisms of rcinomas , lung adrcinomas , and chircinomas . MET ampn cancer , 20, 21.n cancer , 23, 24.n cancer , 26. HowTherefore, we systematically assessed the molecular status of MET in ccRCC in correlation with clinical features in a large, hospital-based series with long-term follow-up information and show that elevated HGFR expression and MET amplifications are evident in a substantial percentage of metastatic and/or aggressive ccRCCs and emerge de novo in RCC metastasis.in-situ-Hybridisation (CISH) were performed on tissue microarrays (TMA) containing tumor and corresponding normal renal tissue from 932 patients with renal cell carcinomas. The study is focused on 763 clear-cell RCC. In total 572 cases could be successfully scored for both, HGFR expression by immunohistochemistry and MET copy number by CISH analysis. The remaining cases were excluded from further analyses either because of insufficient tumor tissue, poor tissue preservation or missing patient information. Figure MET CISH results. The clinical and pathological features of the study population are summarized in Table Immunostains and Chromogenic-MET copy number gains could be detected in 310 cases, 47% (272/572) tumors exhibited >2-4 MET copies per nucleus and another 7% (38/572) tumors more than 4 copies per nucleus, the remaining 46% (262/572) cases showed 2 copies per nucleus. Increase of MET/centromer-7 ratio > 2 was only seen in 7 tumors, all being positive by immunohistochemistry; furthermore 37 out of 38 tumors with more than 4 copies per nucleus were positive by HGFR immunohistochemistry  2 MET copies/nucleus. However, the protein levels of HGFR and MET copy number showed a weak positive correlation , 51% (292/572) showed low (defined as immunoreactive score (IRS) < 6) and 32% (184/572) high cytoplasmic positivity (defined as IRS ≥ 6), the remaining 96 cases were completely negative (17%). hemistry +B. In tuMET copy number gains in 5 out of these 6 cases with an average of two MET copies per nucleus in the primary tumor and an average of 4.4 MET copies per nucleus in the metastasis. The remaining case showed stable MET copy numbers in primary tumor and metastasis.As MET signaling has been reported to be pivotal in development of cancer and metastasis  we creatMET copy number gains, HGFR overexpression has to be based on other mechanisms than amplification. As mutations that inactivate the Cbl binding site lead to constitutive HGFR expression [MET mutations by targeted next-generation sequencing in 9 tumors with high HGFR protein levels and 2 MET copies/nucleus and 2 tumors with high HGFR protein levels and gains of MET copy numbers. No MET mutations were found using Ion AmpliSeqTM Cancer Hotspot Panel v2 comprising exon 2, 11, 14, 16, and 19 of the MET gene. Besides VHL mutations, we did not identify other recurrent mutations in the examined genes.As increased HGFR protein levels were observed in the absence of pression , we checThe proportion of tumors positive for HGFR by immunohistochemistry increased with dedifferentiation (P < 0.001) and occurrence of distant metastasis (P = 0.049) Figure . For exaMET copy number gains were accompanied by dedifferentiation (P < 0.001), higher tumor extent (P < 0.001), positive lymphnode status (P = 0.006), and occurrence of distant metastasis (P = 0.02) . Whereas more than half of T1-carcinomas (54%) showed two MET copy numbers/nucleus, tumors without copy number gains represent only 18% of locally advanced (T4) carcinomas. No consistent association between MET copy numbers and patient age was observed.) Figure . For insThe median time of follow-up was 8 years , among the 572 patients, 189 had died from ccRCC by the end of the follow-up.MET copy numbers per nucleus, cases with more than two MET copies exhibited significant shorter cancer-specific survival (P = 0.001) and time to progression (P = 0.046). Kaplan-Meier plots are depicted in Figure When tumors were grouped according to HGFR expression, univariate survival analysis revealed a decrease in cancer-specific survival (P = 0.008) in patients affected by tumors with high HGFR expression compared to tumors with low/no HGFR expression. In contrast, no consistent relationship was observed between HGFR expression and time to progression (TTP). When tumors were grouped based on n P = 0.06. KaplanDifferences in cancer-specific survival or time to progression did not remain statistically significant after adjustment for established prognostic factors in the multiple regression analysis Table .MET oncogene is a validated therapeutic target for a number of malignancies and results obtained from clinical trials are encouraging [Despite comprehensive translational research efforts in renal cell carcinoma and availability of targeted therapy options, at present neither prognostic nor predictive biomarkers are established for routine clinical treatment stratification . Indepenouraging .MET copy number gains with dedifferentiation, tumor extent, lymph node, and distant metastasis. Limitations of this study are the retrospective single-institution design and the use of tissue micro array technique, which enables studies on large collectives, but may disregard tumor heterogeneity. However, our findings are in accordance with previous reports showing that elevated HGFR expression correlates with worse cancer-specific survival [MET copy number gains correlate with unfavorable patient outcome. In addition, a recent study on brain metastasis of ccRCC indicated MET overexpression as an independent prognostic factor for brain metastasis-specific survival [Our results demonstrate that elevated HGFR protein levels are associated with dedifferentiation and distant metastasis and survival . Moreovesurvival .MET copy number status was not determined. Schoffski et al. reported that ALK/ROS/MET inhibitor Crizotinib induced long lasting disease control in metastatic papillary renal cell carcinoma type 1 with MET mutation and long term stable disease in a case with MET amplification. In contrast, among patients with papRCCs devoid of MET amplification or mutation, none achieved a response upon Crizotinib treatment [At present, reliable data regarding diagnostic criteria and predictive cut offs for anti-MET treatment are not available for ccRCC, thus further studies have to define applicable parameters. Recently published final results of the phase 3 trial METEOR comparing cabozantinib  versus everolimus in patients with advanced renal cell carcinoma  do not preatment .MET amplification. Hence, the study was unable to confirm MET status as a predictive biomarker [The MET pathway is associated with breast cancer progression and anti-Met therapies are currently evaluated in breast cancer patients , 35, 36.iomarker .MET amplification seems to be predictive based on recently reported results of a Crizotinib phase I study [In non-small cell lung cancer the results are inconsistent , however I study .MET copy number assessment or examination of HGFR protein expression is the approach of choice to identify potential therapy responder. Due to discrepancies in protein levels and copy number status in a subset of tumors, we recommend to perform both tests, in situ hybridization and immunohistochemistry, until reliable data is available.At present it remains unclear, if MET copy number gains (>4/nucleus) occur in 7% of therapy-naive ccRCC. Importantly, this proportion increases to 20% in patients with high-grade carcinomas. Naturally, tumor spreading and consecutive need for systemic therapy is more likely in patients with high-grade ccRCC. Furthermore, we identified elevated HGFR expression in 6 and MET copy number increase in 5 metastases out of 15 ccRCCs without elevated expression of HGFR or copy number gains of MET in the primary tumor. These findings indicate that the MET pathway may be a promising target especially in ccRCC patients with high grade or metastasized disease and the MET status should be re-evaluated in recurrences or metastasis.Our results reveal that MET is deregulated in a subset of ccRCC and MET is transcriptionally activated by hypoxia and acts as mediator of antiangiogenic therapy resistance in models of solid tumors [MET amplification is seen in therapy resistant RCCs, resembling a resistance mechanism evident in lung cancers with EGFR activating mutations treated with Gefitinib [d tumors , 45. Impd tumors . Therefoefitinib .MET copy number gains in the context of metastatic spread and initial in-vitro data indicating that MET-signaling acts as mediator of antiangiogenic therapy [Association with clinical and pathological features in primary tumor,  therapy , suggestSeveral compounds including small-molecule TKIs, monoclonal antibodies, and anti-HGF compounds are available for selective inhibition of MET signaling , 49 or iMET copy numbers should not only be assessed in the primary tumor, but also, maybe even more important, in recurrences or metastasis to guide anti-MET therapy in ccRCC patients.In conclusion, our findings highlight inhibition of MET signaling pathway as a promising new target for the treatment of ccRCC patients and prognostic significance of the molecular status of MET. Furthermore, HGFR expression and Tissue samples from 932 patients with primary renal cell carcinomas treated at the Department of Urology at the University of Heidelberg between 1987 and 2005 were collected. The human tissue samples were provided by the Tumour Tissue Bank of the National Centre for Tumour Diseases Heidelberg after approval by the Ethics Committee of the University of Heidelberg. As described previously, clinical follow-up was available for 911 patients, who were prospectively evaluated every 3 months for the first 2 years after treatment, every 6 months for the next 3 years, and yearly thereafter (chest x-ray or thoracic computed tomographic (CT) scan; abdominal sonography or CT scan or magnetic resonance imaging; serum chemistry). No adjuvant treatment of localized disease was administered. Patients with metastasized disease and with a Karnofsky performance index of ≥80 and no medical contraindications received palliative interferon-alpha– and IL-2–based immunotherapy. No targeted therapeutic approaches were performed .A tissue microarray containing 932 primary tumor and corresponding normal tissue samples of 932 patients was composed. The tumors were graded according to the three-tiered nuclear grading system recommended by the WHO Classification of Tumours 2004 and pathologically staged based on the TNM classification (2009). In total, a set of 19 array blocks was generated, each containing 200 tissue core specimens, representing 50 patients per array. A morphologically representative region was chosen from each of the renal cell carcinomas and two cylindrical core tissue specimens per tumor block measuring 0.6 mm in diameter were punched from these regions and arrayed into the recipient paraffin block. Further details have been described previously .In addition, together with the Tumor Tissue Bank of the National Centre for Tumor Diseases Heidelberg, a second tissue microarray was composed containing tumor tissue of 18 patients with metastasized RCCs. Briefly, representative regions of the primary tumor and the matching metastasis was compiled; three  cylindrical cores (diameter 1 mm) were punched from the donor blocks and arrayed into the recipient paraffin block using a semiautomatic system . The origins of metastases were adrenal gland (2x), bone (2x) and lymph node (14x).After heat-induced antigen retrieval using the target retrieval solution ULTRA Cell Conditioning  tissue microarray slides were stained with an anti-total c-MET (SP44) rabbit monoclonal primary antibody . Staining was performed using an automated staining system BenchMark ULTRA  in accordance with the manufacturer's instructions, the following solutions were used: OptiView DAB IHC Detection Kit (760-700), Hematoxylin I (790-2208), Bluing Reagent (760-2037). The arrays were independently scored by two pathologists (S.M.-G. and W.R.) blinded to tissue annotations and patient outcomes. For the immunohistochemical semiquantitative assessment of HGFR expression, the product of the scores of staining intensity and quantity of immunoreactive tumor cells was calculated based on the following scoring system: the intensity ranged from 0 = negative, 1 = low, 2 = medium to 3 = high; the quantity comprised 0 = no expression, 1 < 10% of positive cells, 2 = positivity in 10% to 50%, 3 = positivity in 51% to 80%, and 4 = positivity in more than 80%. The final immunoreactive score (IRS) score (ranging from 0 to 12) is obtained by multiplication of the intensity score and the quantity score.MET gene at 7q31 has been used according to the manufacturer's instructions.The commercial available Probe  a mixture of a Dinitrophenyl-labeled CEN 7 probe specific for the alpha satellite centromeric region of chromosome 7 (D7Z1) and a Digoxigenin-labeled probe specific for the Mutation analysis was performed as described in detail previously . BrieflyData analysis was performed using the Ion Torrent Suite Software (version 4.4). After base calling, the reads were aligned against the human genome (hg19) using the TMAP algorithm within the Torrent Suite. Variant calling was performed with the variant caller plugin within the Torrent Suite Software and the IonReporter package using a corresponding bed-file containing the coordinates of the amplified regions. Only variants with an allele frequency > 5% and minimum coverage > 100 reads were taken into account. Variant annotation was performed using Annovar . AnnotatMET copy number increase was first assessed by log-rank tests and represented using Kaplan-Meier plots. In order to account for the influence of established prognostic factors, hazard ratios (HRs) and 95% confidence intervals (CIs) were adjusted for patient gender and age, tumor extent, lymph node metastasis, distant metastasis, grade of malignancy, and ECOG Performance Status in a multiple Cox proportional hazard regression. Data were analysed using the R software package (https://cran.r-project.org/). For count data, Fisher's exact test (two-sided) was used. Spearman's rank correlation was estimated to quantify the relationship between MET copy numbers and HGFR expression. Probability values <0.05 were considered to indicate a statistically significant result.Survival was calculated from the date of nephrectomy to two different events: cancer-specific survival  and time to progression . Association between survival times and HGFR expression /


Concrete is one of the most popular construction materials in the world. Chemical admixtures are ingredients added to concrete to enhance its properties. However, most chemical admixtures on the market today are expensive, thereby making them out of reach for small consumers of concrete. In Africa, use of chemical admixtures is rare despite the harsh weather conditions. In the current study, Gum from Acacia karroo (GAK) was used as a water-reducing admixture in concrete. A slump test, density and compressive strength were studied using different dosages of GAK while neat concrete was the control. Results showed that slump increased by 200% at a 2% dosage of GAK. This enabled reduction of water-to-binder (w/b) ratio from 0.61 to 0.48 for samples with a 3% dosage. Reduction in w/b resulted in increased compressive strength of 37.03% above the control after 180 days of curing for a 3% dosage. XRD studies also showed a decreased rate of hydration in the presence of GAK in concrete. It was concluded that GAK can be used in concrete as a water-reducing admixture, which is environmentally-friendly, thus producing sustainable and greener concrete. Concrete is a composite material made up of cement, aggregates, water, chemical admixtures and mineral admixtures. Use of chemical admixtures has grown considerably in the last four decades . Althouget al. [et al. [Most admixtures are either organic or inorganic. Among the organic chemical admixtures used in concrete are natural gums. Gum arabic and tragacanth gum, which are natural gums, are good deflocculants, as well as good thickeners. They may have water-reducing properties and a dispersing effect and have been used as pumping aids . These get al.  and Rols [et al. . The mec [et al.  classifi [et al. . Howeveret al. [GAK comes from Acacia karroo Haynes, which grows mainly in the southern countries of Africa , while gum arabic (GA) comes from Acacia senegal or seyal species, which grow in countries in the north of Africa, like Sudan, Chad and Nigeria. Gum arabic is mostly referred to as gum from Acacia senegal and seyal and is mostly sought because of its purity and high grade . GA is uet al.  reportedet al.  used it Both gums (GAK and GA) contain different proportions of neutral sugars. They are made up of side chains of D-glucuronic acid with L-arabinose terminal units or L-rhamnose with a backbone made up of D-galactose units . Gum AcaAfrica needs new a approach to the use of admixtures that have specific modification properties of concrete in addition to being compatible with African climatic conditions. For example, when concrete is mixed at elevated temperatures, there is a quick loss of workability due to high evaporation of mixing water, and the tendency is to add more water to the mix. Chemical admixtures can be used to increase workability without increasing the amount of mixing water. Thus, there is a need to develop admixtures that are natural, inexpensive and environmentally friendly. In this study, the influence of GAK on the mechanical properties of concrete was investigated. Concrete performance was determined by conducting a slump test, air entrainment and compressive strength at different water-to-binder(w/b) ratios and different GAK dosages. X-ray diffraction (XRD) analysis was carried out just to give an idea of hydration at 56 days.Gum Acacia karroo (GAK) was picked from Pretoria Botanical Garden. The tears were hand-picked from the bark , cleanedOrdinary Portland cement CEM I (grade 52.5 MPa) blended with 25% unclassified fly ash was used to prepare all concrete specimens. All of the cement and fly ash was donated by Pretoria Portland Cement (PPC). X-ray fluorescence (XRF) was used to determine the composition of cement and the composition of fly ash. Potable water was used for mixing.Crusher sand (CS) and coarse aggregates (CA) were both donated by Rosslyn Quarry in Pretoria. The fine aggregate conformed to SANS1083: 2006 . The coaThe fineness modulus (FM) of sand was 3.2, which fell within 2.9–3.5, which is categorized as coarse sand fineness . GradingThe slump test was carried out according to SANS 5863-3: 2006  immediatConcrete density was measured at the age of testing, by weighing all of the specimens after curing, using an electronic weighing balance before pressing the cubes for the compressive strength test. The density of each specimen was calculated by dividing the weight of the cubes by the volume of molds.The pressure method was used to measure the air content in concrete as per EN 12350-7: 2009 .The compressive strength test was carried out according to SANS 5863: 2006 . The deset al. [Accelerated carbonation was carried out in a carbonation chamber as per the method previously used by Gonen et al. . The samα radiation. The phases were identified using X’Pert Highscore plus software. The relative phase amounts (weight %) were estimated using the Rietveld method (X’Pert Highscore Plus software).Four samples with high dosages of GAK  were prepared for XRD analysis. The aim of this analysis was to identify phases of hydration products at high dosages. The samples were analyzed with a PANalytical Empyrean diffractometer with a PIXcel detector and fixed slits with Fe filtered Co-KPrecipitate from reagents for testing GAK contained 70% sugars, like fructose, glucose and sucrose, and its pH in soluble form was 4.5.Slump test results for concrete with different dosages of GAK are shown in As the dosage increased, the effect of deflocculation of cement grains by GAK is seen by increased slump values. Increased fluidity in concrete in the presence of GAK can be explained by the fact that cement particles tend to form clusters, which trap water within them in the absence of water reducers. An increase in the fluidity of concrete in the presence of high dosages of GAK could be due to the break up of the clusters and maintaining dispersion, so that each particle exists as an individual. This results in increased mobility of the fresh concrete . FluiditMixes with GAK and a reduced w/b ratio lost slump after approximately 10 min of mixing, thereby requiring more effort for compaction and reducing the compaction time. This is due to the adhesive nature of GAK, which made the particles stick together, resulting in a stiff concrete mix. Loss of slump could have been probably caused by the amount of gypsum and the tri-calcium-aluminate in cement . This caValues of mean density (plus their standard deviation) of concrete samples with different dosages of GAK are shown in 3 .et al. [et al. [Most polysaccharide gums entrain air according to Wada et al. , Chandra [et al.  and Lazi [et al. . This waDifferent dosages of GAK did not change the amount of air entrained significantly. This result was not expected to a certain extent due to the fact that there was a substantial decrease in strength with the increase in dosage. According to , some adCompressive strength results at different curing ages and different GAK dosages are shown in Comparison of the mean compressive strength of the control with the compressive strength of samples with a reduced w/b ratio showed an increase in strength see , which iAt a 1% dosage, the compressive strength with a w/b of 0.56 was less than that of the control at 7, 28 and 56 days, but higher by 4.3% than the control at 180 days. This phenomena could be explained by the reduced hydration rate at an early age due to the presence of GAK. The highest compressive strength was achieved at a 3% dosage of GAK with a w/b ratio of 0.48 relative to the control. Such high strength concrete is usually achieved by increasing the amount of cement. In this research, high strength concrete was achieved by using the same cement content, but reducing the w/b ratio by reducing the water content.The water-binderratio has a critical impact on the concrete strength characteristic. For the proper chemical reaction to take place, minimum water (about 3% of mixing water) is required, while extra water increases the slump and reduces the strength. Excess water produces concrete with higher capillary porosity. Thus, water-reducing admixtures release the water trapped within the cement grains, thereby availing more water to participate in hydration. In the construction industry, a low w/b ratio is used to achieve concrete with high strength and high quality. However, this is not achievable without the use of admixtures if concrete is to remain workable, free from cracking and excessive shrinkage. Therefore, use of GAK as a natural plasticizer is beneficial to the construction industry, where high performance concrete is required. The increased compressive strength of samples with a reduced w/b ratio was as a result of the water reducing power of GAK while keeping concrete workable.Concrete with a reduced w/b ratio develops strength at a higher rate than concrete at a higher w/b ratio. This is an important factor, since it is possible to remove formwork earlier, saving time during construction. The other important benefit is saving cement while achieving concrete of high strength. In most cases, when concrete of high strength is required, cement content is increased. Thus, reducing the amount of cement used is important for the environment, as well as reducing the cost of concrete.The carbonated part of the specimen had no coloration, while the carbonated part turned purple due to the high alkalinity . It was The carbonation rate depends on the concentration of CaOFurthermore, the entrained air could also play a role in carbonation by increasing the transportation mode of the gas. The increase in carbonation depth could have been associated with less denser and lighter concrete. These results were confirmed by reduced compressive strength after carbonation, as shown in XRD studies were carried out on samples after 56 days of curing. The control, the sample with a 1% dosage and a w/b ratio of 0.61 and two samples containing 2% and 3%, but with a w/b ratio of 0.52 and 0.48, were studied. The studies revealed almost a similar pattern for all samples with and without GAK, but with different intensities, as shown in The delayed hydration observed was as a result of calcium ions forming glucosate and sucroate in the presence of glucose and sucrose, respectively . The forn cement .(1)In hot weather climates, there is a tendency to add water to concrete to make it more workable. The addition of GAK to concrete can reduce this tendency. There was a remarkable improvement of concrete workability when GAK was used at above a 0.5% dosage. At a 2% dosage, the slump of concrete increased by 200%. However, the drawback observed was the quick slump loss due to the adhesive nature of GAK. This type of concrete is applicable in pre-cast technology.(2)The increase in GAK dosage reduced the compressive strength and density of concrete. The air content also increased with increased dosage, which could have contributed to the compressive strength reduction. The highest decrease in compressive strength observed was 24.5% lower than the control at a 0.5% GAK dosage. In the case of a reduced w/b ratio from 0.61 to 0.48, the highest strength was observed at 3% GAK at all ages. The strength gain at 180 days was 37.03% higher than the control. The use of GAK as a water-reducing admixture is a possibility. Increased strength means GAK can be used as a water-reducing admixture, resulting in the reduction of cement demand, which in turn reduces carbon emissions and energy used, thereby providing the construction industry with a cleaner, greener and environmentally-friendly admixture. In addition, the gain of strength at an early age means formwork can be removed earlier, thereby decreasing construction time.(3)The carbonation depth of concrete slightly increased with increased GAK dosage. The highest carbonation depth increased by 22.8% at a 1% dosage above the control. Samples with a reduced w/b ratio showed a decrease in carbonation.(4)The XRD analysis gave an idea of the effect of GAK on hydration. The addition of GAK at higher dosages enables lower w/b ratios. This, in turn, showed an improved hydration rate relative to the control.The effect of GAK use in concrete was tested, and different properties were studied both in the fresh and the hardened state. From the results obtained, the following conclusions were made:


Photobacterium halotolerans strain MELD1 was isolated from mercury and dioxin contaminated site from Tainan, Taiwan. It has been shown to reduce Hg2+ to Hg0. The 4,758,027 bp genome of P. halotolerans MELD1 has a G + C content of 50.88 % and contains 4198 protein-coding and 106 RNA genes. Genomic analysis revealed the presence of a number of interesting gene cluster that maybe involved in heavy metal resistance, rhizosphere competence and colonization of the host plant.Mercury impacts the function and development of the central nervous system in both humans and wildlife by being a potent neurotoxin. Microbial bioremediation is an important means of remediation of mercury-contaminated soil. The rhizospheric The online version of this article (doi:10.1186/s40793-016-0177-3) contains supplementary material, which is available to authorized users. Photobacterium genus are Gram-negative bacteria belonging to the family of Vibrionaceae [Photobacterium halotolerans was first reported by Rivas et al. [Species of the ionaceae  and has ionaceae  or symbiionaceae . Photobas et al.  which waPhotobacterium halotolerans MELD1, a plant growth promoting gamma-proteobacterium that was isolated from the root of Phragmites communis Trin. Ohwi [mer operon, consisting of mercury reductase gene (merA) that helped in the conversion of Hg+2 to Hg0 [P. halotolerans MELD1 along with its annotation for rhizosphere competence, plant growth promoting and heavy metal resistant genes.In our previous study we isolated in. Ohwi , a large2 to Hg0 . It was Photobacterium halotolerans MACL01T as compared to other Photobacterium strains. Classification and general features of P. halotolerans MELD1 are shown in Table The genes encoding for the 16S rRNA were amplified by PCR using two universal primers, E8F and U1510R followed by BLAST against NCBI 16S rRNA sequences database. The 16S rRNA sequences of MELD1 and closely related strains were aligned by ClustalW and minimized with BioEdit . The phylogenetic tree was generated by One Click phylogeny analysis on Methodes et Algorithmes pour la Bio-informatique Lirmm website  and expoP. halotolerans MELD1 could grow at 6 % NaCl as compared to P. halotolerans MACL01T which can grow at 8 % NaCl [Strain MELD1 is a Gram-negative bacterium, rod-shaped and motile by means of polar flagella. They are usually 2–4 μm in diameter . One microgram sonicated DNA was end-repaired, A-tailed and adaptor-ligated following the Illumina Trueseq DNA preparation protocol.DNA was sequenced using Illumina Solexa technology. Ten microgram of total DNA was sonicated by Misonix 3000 sonicator to the size ranging from 400 to 500 bp. The genome size was estimated prior to assembly using Bioanalyzer de novo using Abyss [ab initio gene prediction algorithms with homology based methods.ConDeTri  was implng Abyss . The genMELD1 genome contained 62 contigs with a size of 4,758,037 bp. The G + C content was 50.90 %  are found to be in the MELD1 genome. Analysis of GC content of MELD1 genome (51 %) portrays that the gene that appear to have a GC content close to that of MELD1 genome, could have been integrated into MELD1 genome through evolution by the process of horizontal gene transfer (Additional file Many bacteria acquire iron from the environment by secreting small iron-binding molecules called siderophores. rganisms . The prerganisms . Similarella spp , P. haloectABC cluster responsible for synthesis and accumulation of ectoine (Additional file Analysis of genome revealed that MELD1 has a number of gene reported to play a role in osmotolerance like glycine-betaine and ectoine. The genome analysis of MELD1 revealed genes involved in glycine betaine synthesis that help MELD1 to maintain osmotic balance in hyper saline environment. It was observed that MELD1 was able to grow at a salt concentration of up to 6 %. It carries an Plants utilize a variety of defense mechanisms against various pathogens, including the production of ROS, hydrogen peroxide , 21. PricheY, cheW, cheA, cheR and cheX (Additional file flg and fli genes responsible for flagella biosynthesis and motility (Additional file xerD, a site recombinase critical for the plant growth promoting rhizobacteria Psuedomonas fluorescens F113 to be an effective rhizosphere colonizer [Azospirillum lipoferum [. MELD1 has quorum-sensing-regulatory genes like luxR and luxU, encodes AI-2 which is implicated in the regulation of biofilm formation and motility [Adhension to the root in endophytic and rhizobacteria is mediated by cell surface structures such as polysaccharides, pili and adhesion . It alsoolonizer . Quorum-ipoferum  modulateipoferum . MELD1 hmotility . Some otmotility , 30 are Photobacterium halotolerans MELD1, a strain having mercury reductase activity has been deposited at NCBI under the accession number JWYV00000000. The version described in this study is the first version, JWYV01000000. MELD1 also contained a cluster of gene’s responsible for heavy metal resistance, heavy metal efflux pumps, antimicrobial compounds, stress resistant, motility, and plant growth promoting genes, which all prove that they can function as a rhizosphere or an endophytic bacteria in a toxic environment. The detailed genome announcement can give insight into the adaption of a marine dwelling bacterium as a terrestrial dwelling endophytic or rhizosphere bacterium and in future might aid in the bioremediation of mercury. Further more extensive research need to be done using molecular techniques to establish horizontal gene transfer in MELD1 with donor species.The 4.7 Mb draft genome of


In many low- and middle-income countries (LMICs), a poor link between antibiotic policies and practices exists. Numerous contextual factors may influence the degree of antibiotic access, appropriateness of antibiotic provision, and actual use in communities. Therefore, improving appropriateness of antibiotic use in different communities in LMICs probably requires interventions tailored to the setting of interest, accounting for cultural context. Here we present the ABACUS study (AntiBiotic ACcess and USe), which employs a unique approach and infrastructure, enabling quantitative validation, contextualization of determinants, and cross-continent comparisons of antibiotic access and use. The community infrastructure for this study is the INDEPTH-Network , which facilitates health and population research through an established health and demographic surveillance system. After an initial round of formative qualitative research with community members and antibiotic suppliers in three African and three Asian countries, household surveys will assess the appropriateness of antibiotic access, provision and use. Results from this sample will be validated against a systematically conducted inventory of suppliers. All potential antibiotic suppliers will be mapped and characterized. Subsequently, their supply of antibiotics to the community will be measured through customer exit interviews, which tend to be more reliable than bulk purchase or sales data. Discrepancies identified between reported and observed antibiotic practices will be investigated in further qualitative interviews. Amartya Sen’s Capability Approach will be employed to identify the conversion factors that determine whether or not, and the extent to which appropriate provision of antibiotics may lead to appropriate access and use of antibiotics. Currently, the study is ongoing and expected to conclude by 2019. ABACUS will provide important new insights into antibiotic practices in LMICs to inform social interventions aimed at promoting optimal antibiotic use, thereby preserving antibiotic effectiveness. Improving the appropriate use of antibiotics necessitates understanding their supply, as well as the social and cultural factors that create demand in the community. As different LMIC settings show distinct rates of over-the-counter antibiotic dispensing4, it is clear that the social, cultural and policy-related determinants of antibiotic practices are context-specific.Antibiotic resistance makes global dedication to facilitate the appropriate use of antibiotics an imperative. Two recent reports on antibiotic resistance have illustrated the importance of conducting studies in low- and middle-income countries (LMICs) where local data are scarce and the problem is significantThe aim of this project is to compare community-based antibiotic access and consumption practices across communities in LMICs in Asia  and Africa , in order to inform the design of, and identify targets for community-based intervention strategies that may be used to promote appropriate antibiotic use. Our hypothesis is that both antibiotic practices as well as their determinants differ across LMICs, and therefore warrant tailored intervention strategies. To examine this hypothesis, we will compare antibiotic access and use in the community between six different LMICs, and identify their main drivers through both qualitative and quantitative measures. A core asset of this study is that it covers multiple aspects involved in understanding community antibiotic practices: supply and demand; reported and observed practices; and local to cross-continental comparison .http://www.indepth-network.org/member-centres). INDEPTH is a global network of Health and Demographic Surveillance Sites (HDSSs), with 45 member centres and 52 HDSS field sites in 20 LMICs in Africa, Asia and Oceania. We will first perform in-depth qualitative interviews and focus group discussions (FGDs) in each participating site to explore the prevailing factors that affect antibiotic practices at each study site. The interviewees will include both antibiotics suppliers and consumers. This qualitative information will facilitate the development of a longitudinal quantitative household survey. This will then be followed by another set of in-depth interviews and FGDs with the aim of explaining any potential discrepancies between reported and observed use of antibiotics within each study site. The supply of antibiotics to the communities involved will be determined by standardized drug outlet inventories and customer exit interviews. The gained insight into antibiotic practices in different LMICs will inform the design of tailored intervention strategies to promote appropriate antibiotic use in the different settings. In addition, this project provides a uniform framework for appraising current antibiotic use patterns, which may subsequently be used in other LMIC communities.The study will be conducted in seven existing health and demographic surveillance system sites in six LMICs  that are part of the INDEPTH-Network 6. Qualitatively exploring individual values and contextual constraints is elemental to the framework.In our case, the resource of interest is the appropriate provision of antibiotics. However, we recognize that simply having appropriate provision does not necessarily translate into appropriate use. There are a number of factors, ranging from individual characteristics, to broader economic constraints, cultural norms, social frameworks, and political structures that can influence how antibiotics can be used. In Sen's terms, these are the 'conversion factors' that enable a possible set of outcomes (what he terms as 'capabilities'). Operationalising the CA is most suitably done by employing both qualitative and quantitative approachesThis is an observational multicentre study that implements a set of interviews among drug suppliers and community members across six LMICs, which will be performed consecutively over a 2.5-year study period. The study consists of two major components that are based on ‘reported experiences’ and ‘observed metrics’, respectively .Initially, antibiotic resources will be ascertained by mapping all antibiotic suppliers  and by phttps://datahelpdesk.worldbank.org/knowledgebase/articles/906519), we selected: Bangladesh and Mozambique representing lower income countries (LIC), Vietnam and Ghana representing lower-middle income countries (MIC-L), and Thailand and South Africa representing upper middle income countries (MIC-U). To facilitate the study, the chosen countries all contain at least one INDEPTH community-based study site. INDEPTH can provide a readily available and relevant sampling frame, and is therefore uniquely positioned to answer pressing questions on health in community settings. Globally, the INDEPTH Network surveillance sites study the life events of approximately 3 million people through on-going demographic surveillance. The network was set up in 1998 and has developed tools to measure, map and track the socio-demographic impact of cause-specific morbidity and mortality in LMIC populations using comparable methodologies in different countries8.The project will take place in rural communities of six countries in Africa and Asia that have been chosen based on their income status in 2013. Using the World Bank classification of LMIC (criteria for 2014)  will be mapped and are eligible to participate in this study. Potential dispensing points will be identified based on both official lists provided by local authorities and local knowledge of the HDSS sites. This includes any formal or informal antibiotic supplier: from public hospital pharmacy to street vendor. The expected number of daily antibiotic encounters (a customer encounter that involves supply of antibiotics), and the proportions of the different types of suppliers will be determined from the mapping exercise.Antibiotic suppliers 18 years old and older are eligible for three of the study elements: the customer exit survey, the inventory of antibiotic resources at vendor level, and the qualitative in-depth interview.In each study site, 20 suppliers will be selected for customer exit interviews. The antibiotic suppliers will be recruited according to highest rank in terms of the number of daily antibiotic encounters.The selection process of 10 antibiotic suppliers for inventories and 16 antibiotic suppliers for exploratory in-depth interviews will ensure similar proportions of the supplier types as identified in the mapping exercise in the corresponding study site. The HDSS field workers will characterized all identified antibiotic suppliers and will calculated the proportions of different types of antibiotic suppliers across the study sites. The suppliers will be randomly selected using the Excel random number function. The randomly selected suppliers will be approached sequentially to get permission to participate in these procedures until the required number is reached. For the qualitative in-depth interviews, employees who interact with customers will be included. Ideally, each in-depth interviewee will be affiliated with a unique antibiotic supplier, but if fewer than 16 eligible antibiotic suppliers can be identified in each site, a maximum of two employees from the same business may participate in the in-depth interviews.Any supplier who does not consent for participation in a particular study element, will still be eligible for inclusion in the other study elements.Community members for qualitative in-depth interview. Community members 18 years-old and older who are willing to speak about their experiences with, and their attitudes towards medicines, are eligible to participate in this study. Different target populations from the community will be included in the initial round of exploratory in-depth interviews. From the HDSS-database, we will select eight mothers who care for children under 5 years old. The other eight participants should not be mothers of children under 5 years old, and they will include two males and two females 18 to 60 years old, plus two males and two females above 60 years old. These participants will be randomly selected against above inclusion criteria. The rationale for this inclusion criteria is that involving subjects from different age categories and gender will assure achieving a more informative view. In addition, children under five years-old and adults above 60 years-old have a higher frequency of healthcare seeking. Household female members, particularly mothers, play a crucial role in managing childhood and family illness, thus they will predominantly be selected. In this study, we will avoid having two participants from the same household  require participants from outside the groups mentioned above, the inclusion criteria will be redefined for that site.Households for longitudinal survey. Households that already participate in the HDSS and have previously provided consent, are eligible to participate in this add-on study at two consecutive time points. An adult household representative (18 years or older) must provide additional written informed consent before the household can be included in the current add-on study. In each study site, 1,000 households will be recruited to participate in this add-on study.Customers for exit interview. At the selected antibiotic suppliers, each customer who leaves the drug outlet will be approached, and asked whether antibiotics were supplied to him/her. In case of an antibiotic encounter, the customer will be asked to participate in the customer exit interview. Recruitment of participants will be stopped whatever comes first, the end of that particular day or 30 antibiotic encounters.To be included in the study, customers have to accept disclosing whether antibiotics were supplied to them and to consent to participate in an interview. Refusal cases will be registered.Sample size HDSS household survey. This is the first study that will compare determinants of antibiotic use among LMICs through Sen’s Capability Approach. The main study outcome is the appropriate use of antibiotics and its determinants. In this study, the provision of antibiotics is considered to be appropriate if all of the following conditions are met: presence of original packaging, before expiration date, supplied with prescription, and with proper indications for use10. If any of these conditions is not met, the provision of antibiotics is considered to be inappropriate. This will be assessed by logistic regression analysis in each study site. In previous studies, antibiotics were supplied to outpatients with a prescription, which is an important criterion in our definition of appropriate use, in 60% of cases in Ghana11, in 50% of cases in Bangladesh12, but only in 10% of cases in Vietnam4. Antibiotics were appropriately dispensed in 20% of pharmacies in Thailand, under criteria stricter than applied in the current study13. Therefore, it is expected that on average 30% of antibiotic use will be classified as appropriate. Based on previous experience with surveys in the participating study areas, a loss to follow-up of 5–10% is to be expected. To avoid the need for subsequent recruitment of extra ‘replacement’ households, 1,100 households will initially be recruited , of which we expect at least 1,000 to participate in two consecutive surveys. Despite the use of show-cards, probably not all household interviewees will be informed about the antibiotic consumption by the household. If 750 HDSS household representatives (representing 3000 HDSS household members) can fully participate, and 20% of household members have an antibiotic intake in the months of survey, we may expect 180 appropriate antibiotic events. Given the rule of thumb of 10 events for each variable in regression analysis, 180 events would allow for 18 variables. This corresponds with the expected number of possible conversion factors to be yielded from the household questionnaires.Sample size customer exit survey. This study will compare antibiotic supply between LMICs using a standardized method. Therefore, the sample size is based on WHO recommendations14 and on previous experience from customer exit interviews.15. A longitudinal study design is preferred, to account for potential seasonal variation in antibiotic supply16. In addition, although the presence of interviewers could influence suppliers’ behaviour (Hawthorne effect), this effect is likely to diminish over time.According to the WHO/INRUD methodology for investigating drug use in health facilities, a representative sample requires 30 prescribing encounters per facility from 20 facilitiesFor each study site, 20 antibiotic suppliers will be selected for customer exit interviews. At each selected supplier, up to 30 antibiotic encounters will be observed on a single day of survey, amounting to a maximum of 600 antibiotic encounters per study site per round of survey. To account for seasonal variations, this survey will be performed at four time points (i.e. four days of survey) spread over a one-year period, synchronized across all study sites. In total, a maximum of 2,400 antibiotic encounters will be recorded at each study site.19. Accounting for high antibiotic exposure of 43%, given an α of 0.05, the number of antibiotic encounters surveyed by this study will have a power of up to 0.90 to identify a significant difference in antibiotic exposure of 5% between study sites, and a power of up to 0.83 to identify a 10% difference between supplier types within each study site. Because the antibiotic burden will be calculated per type of antibiotic, and it is unknown to what extent different types of antibiotics will be supplied, no reliable power estimation can be given for this second parameter.The main study parameters are antibiotic exposure and antibiotic burden. Previous estimates of antibiotic exposure through customer exit surveys ranged from 21% at private retail pharmacies in India, 29% at drug stores in Mexico, to 39-43% at public/private suppliers in IndiaRationale for other sample sizes. The mapping of antibiotic suppliers will be as exhaustive as possible. The choice of a number of 10 inventories of antibiotic resources per study site is based on the expected minimum number of antibiotic suppliers present in any of the participating study sites. This sample will be sufficiently large to provide a fair representation of antibiotic suppliers. Qualitative investigation of this study topic is quite limited, but experience with qualitative research instruments in the participating study sites indicates that data saturation will be achieved with the number of interviews planned.To assess the level of a participant’s knowledge on antibiotics, the following three instruments will be employed in the HDSS survey and the antibiotic customer exit interview.First, three multiple choice questions (each with four options) will be posed about antibiotics. Second, the HDSS field worker will display a first show-card with photos of three pills that are commonly available in the study area: paracetamol, a non-steroidal anti-inflammatory drug, and an antibiotic. For the development of these show-cards a pharmacist who works in the corresponding country and is knowledgeable about local outpatient antibiotic supplies will be consulted. The respondent will be asked to indicate the antibiotic pill. Third, the HDSS field worker will display a second show-card with photos of five different antibiotic pills that are commonly available in the study area and will confirm that these are in fact antibiotics.If the respondent provides two or more correct answers to the multiple-choice questions, his/her level of knowledge on antibiotics is classified as A. If the respondent provides less than two correct answers to the multiple-choice question, but does correctly indicate the antibiotic pill on the first show-card, his/her level of knowledge on antibiotics is classified as B. If the respondent provides less than two correct answers to the multiple-choice question and does not correctly indicate the antibiotic pill on the first show-card, his/her level of knowledge on antibiotics is classified as C.As mentioned above, the main study parameter of this study component is appropriate use of antibiotics, which is a composite measure. Conversion factors that may affect the relation between appropriate provision of antibiotics and appropriate use of antibiotics collected per study site are:- Sex and age- Education- Socioeconomic status, according to metrics collected by the INDEPTH network HDSS surveillance site- Level of knowledge on antibiotics: A / B / C- The number of episodes of antibiotic intake per person during the months of survey- The proportion of episodes of antibiotics intake that are preceded by a prescription- http://www.kith.no/upload/2705/ICPC-2-English.pdf): chest pain / cough / dental / dyspnoea / ear and eye / fever / gastrointestinal / gynaecological / headache / male genital / musculoskeletal / nose / preventive / skin and soft tissue / surgery related / throat / urinary / wound.The indications for antibiotics’ use as reported by the community members. The indications will be classified to one of the following categories, based upon the International Classification of Primary Care (ICPC-2e v5) - Knowledge of and attitudes towards healthcare, medicines, antibiotics and antibiotic resistance, of both antibiotic suppliers and users- Expectations of and experiences with the local healthcare system- Social and cultural context- Regulatory issues20. The denominator of antibiotic burden is introduced to account for the size of the customer population to which the DDDs are supplied. Here, the denominator is set to 100 customers encounters at the supplier involved on the day of survey. To enable calculation of antibiotic burden, the antibiotic’s name, strength, units and dose will be collected for each antibiotic encounter from the customer. Supplied antibiotics will be recorded by their generic names, and they will subsequently be classified according to the WHO Anatomical Therapeutic Chemical (ATC) classification system20.The main study parameters of this study component are the antibiotic exposure and the antibiotic burden within the community site studied. Antibiotic exposure is expressed as the proportion of customers leaving a supplier to whom antibiotics are supplied. To this end, the number of customer encounters with and without antibiotics will be recorded. Supplied antibiotics formulated in drops or creams are not considered to be an antibiotic encounter. The antibiotic burden is expressed in Defined Daily Doses (DDDs) supplied per 100 customer encounters on the day of survey, for each type of antibiotic. The DDD is the assumed average daily maintenance dose for a drug used for its main indication in adultsAdditional parameters collected in each study site are:- The distribution of supplier types: Suppliers will be classified by legal permit (yes/no), funding (public / private / missionary), and type . In addition, the number of daily antibiotic encounters will be estimated.- The proportion of essential antibiotics that is available and appropriately provided, and their price: Appropriateness of provision will be assessed by storage conditions, original packaging, expiration date, appropriate package insert, presence of prescription and by verbal directions for use.The proportion of antibiotics that is supplied upon prescription.- The distribution of customer types (age and sex) and end user types .- Customers’ level of knowledge on antibiotics: A / B / C.- The indications for use of antibiotics as reported by the customers: The indications will be classified to one of the following categories, based upon the International Classification of Primary Care (ICPC-2e v5): chest pain / cough / dental / dyspnoea / ear and eye / fever / gastrointestinal / gynaecological / headache / male genital / musculoskeletal / nose / preventive / skin and soft tissue / surgery related / throat / urinary / wound / other.Data analyses will be performed at several stages throughout the study. To facilitate uniform data processing, prior to the interviews, a workshop on data analysis will be provided to qualified representatives from each study site. Directly after the mapping of all antibiotic suppliers in a study region is completed (month 6), the rank in number of daily antibiotic encounters and the proportion of each type of antibiotic supplier will be calculated to inform the selection of supplier participants. Alongside the execution of the exploratory qualitative in-depth interviews and FGDs, the qualitative data will already be analyzed locally (month 7 to 10), thereby providing a basis for iteratively refining the design of upcoming interviews, and eventually the design of the quantitative HDSS household surveys. Soon after the first 6 months of the HDSS household survey and customer exit survey have passed (month 18), an interim analysis will be performed. These results will be triangulated with previously obtained qualitative data to design a second round of site-specific in-depth interviews and FGDs. The final seven months of the study will be dedicated to final data processing, analysis, and reporting.5. Verification of the data and themes will be conducted by members of the research team with skills in qualitative analysis. This will involve validating and assessing the trustworthiness of the qualitative data using the four constructs of credibility, transferability, dependability and conformability. The qualitative work will comply with the consolidated criteria for reporting qualitative research (COREQ)6.To analyse the qualitative data, a thematic analytical approach will be employedThe main quantitative parameters will be presented as a comparison between the six study sites. Appropriate use of antibiotics will be presented as a percentage of the reported episodes of antibiotic intake per study site. The relation between appropriate provision  and appropriate use of antibiotics will be presented in a dot plot with each dot representing one study site. Finally, a model will present for each study site that contains the site-specific determinants of appropriate antibiotic use, accompanied by their odds ratios and 95% confidence intervals.Sample size HDSS household survey). True independent determinants of appropriate antibiotic use will be determined by multivariate binary logistic regression analysis using a stepwise backward elimination approach based on the probability of the likelihood-ratio statistic. All tests will be performed in two-sided fashion, and a p-value of <0.05 will be considered statistically significant.Statistical significance of differences in the proportion of antibiotic intake episodes that involves appropriate antibiotic use between study sites, will be tested by Chi square testing using the sum of all registries in each study site. The presence of a statistically significant correlation between appropriate antibiotic provision and appropriate antibiotic use across the six study sites will be assessed using Spearman rank testing. Potential determinants of appropriate antibiotic use will be identified by univariate binary regression analysis. Variables that display an association with appropriate antibiotic use in univariate analysis with a p-value < 0.1 will be selected for multivariate analysis . The antibiotic burden needs to be normalized through calculation, prior to presentation in a summary metric per study site. First, the amount of antibiotics supplied is normalized to the number of Defined Daily Doses (DDDs), according to WHO guidance. Subsequently, the sum of DDDs supplied during the surveyed time frame is normalized for the size of the customer population to which the antibiotics were supplied in that time frame. Our standardized measure of antibiotic burden is the number of DDDs of a given antibiotic per 100 customers. This number will be obtained by taking the sum of DDDs at each supplier (four survey days combined), divided by the total number customer encounters surveyed, and multiplied by 100. The antibiotic burden is a continuous variable that will be presented as an average or median of its distribution among suppliers for each study site .Missing data will be coded as such and accounted for in subsequent data representation and statistical analysis.The data format for statistical testing of differences between study sites matches the data presentation as proposed above. For antibiotic exposure, the sum of all registries in each study site (from 20 suppliers combined) will be compared. For antibiotic burden, its distribution among surveyed suppliers per study site will be compared. Chi square testing will be applied for antibiotic exposure (Fisher exact if <10 cases in any cell), and for antibiotic burden a two-way ANOVA or Kruskal-Wallis test will be performed  for each type of antibiotics. All tests will be performed in two-sided fashion, and a p-value <0.05 will be considered statistically significant.Data from this study will be reported and interpreted at a closing meeting. Subsequently, the results will be disseminated via meetings with stakeholders, conferences, and publications in peer reviewed journals. Authorship and reporting of this work will follow international guidelines.Given that written informed consent is obtained for all study elements, the anonymized data collected in all study elements are eligible for use in future non-related studies.Currently, the study is ongoing at its third phase and will be completed by the end of 2018. The insights gained in relation to antibiotic practices in different LMIC will inform tailored intervention strategies to promote appropriate antibiotic use. In addition, this project provides a uniform framework for appraising current antibiotic use patterns, which may subsequently be used in other LMIC communities.The protocol was reviewed by the Oxford University Tropical Research Ethics Committee , and the local ethical committees of each participating study site including: (1) Vietnam Ministry of Health (MoH) Institutional Review Board (IRB) (6670/QD-BYT); (2) Institute for Population and Social Research (IPSR)-IRB, Mahidol University, Thailand (2016/04-035); (3) Ethical Review Committee (ERC), icddr,b, Bangladesh (PR-16053); (4) Ghana Health Service Ethics Review Committee ; (5) Mozambique Ministry of Health IRB (CIBS-CISM/101/2016); and (6) Human Research Ethics Committee, University of the Witwatersrand, South Africa (M160753).All potential participants will be informed about the study and asked for their consent by trained HDSS field workers. Information about the study will be provided both orally and in writing.Given the real-time design of the customer exit interviews, potential participants will be informed about the study and asked for written consent during the course of one conversation. The local language written study information and informed consent criteria will be read aloud to a potential participant by the HDSS field worker. In addition, customer participants will receive a copy of the written study information. Only after written consent is provided will the interviews be conducted. This is a detailed and well-written protocol paper, the aim of the study being to investigate community antibiotic use in 6 LMICs in Africa and Asia. It seems like a very complex study to coordinate, hence the use and collaboration of the INDEPTH network. That also seems to be the limitation - it is not clear if INDEPTH network sites in these countries differ from those sites where INDEPTH does not operate as many of these are large countries where a variety of antibiotic supply and usage practices may exist in different geographic locations. Nonetheless, this study will advance our knowledge of antibiotic prescription and usage practices in the 6 countries, and I look forward to learning about the results.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Datasets are not relevant to this paper: it is a protocol. This paper describes a protocol that will use six sites in the INDEPTH network to study antimicrobial access and use in resource limited settings. Antibiotic use and distribution patterns will be catalogued following interviews and focus group discussions with suppliers and patients. A longitudinal survey will also be nested in the HDSS questionnaire. Thereafter use will be monitored by mapping and inventorying supply chains.  The study will further investigate discrepancies between use data reported in qualitative surveys and inventoried amounts of antibiotic. Antibiotic use is poorly understood in most non-affluent countries where availability without prescriptions and the existence of robust informal sectors for their distribution pervade. Therefore this is a valuable study that will be of interest to readers who work in or on this type of setting. The study hinges on its good design and conducting the right number and types of interviews in order to garner a realistic picture of antimicrobial use. I found the design adequate for this purpose and especially liked some of its iterative components, which ensure that preconceived ideas, which have a high likelihood of being wrong in this little-studied field, are not the basis for designing key tools for the study. Performing the study at HDSSes helps to ensure appropriate coverage and provide important background information. Additionally, extracting use information using a variety of research methods as the authors plan to do, has the best chance of uncovering unknown or under-recognized facets of antimicrobial use. Given that I am a scientist, I would also suggest social scientist review the methodological components of the protocol as well.  My one critique is connected to a theoretical base for the study. My familiarity with Amartya Sen's Capability Approach is limited but I found it difficult to map onto the antimicrobial use and access problem that the investigators are studying. Unlike other resources that are more directly connected to wealth, needing or using more antibiotics is not necessarily a reflection of wealth or better resources. Improving access is good but only to certain extents and within certain parameters. And the gold standard for antibiotic use would be prescription after susceptibility pattern but access to testing and prescribers are not part of this study. There are economic analogies to the antimicrobial use and resistance paradigms , I am just not sure that this is the right one. I am not sure that this approach adds anything to the study, or if so, what the value of what it adds actually is. If there is value, this could be made clearer.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Amartya Sen’s capability approach, although often used in relation to wealth, need not be limited to economic inputs. The framework allows a more nuanced approach to considering how various inputs do or do not translate to desired outcomes. A simple example to illustrate its utility is the case of food provision during famines. Simply providing food does not necessarily alleviate hunger during a famine. The Capability Approach allows us to understand how people translate the resource, in this case food, to a desired outcome, in this case alleviation from hunger. What we are arguing here with the Capability Approach is that ‘appropriate’ provision of antibiotics may not translate to its ‘appropriate’ use and the factors that affect this disconnect need to be examined. If we assume that antibiotics are prescribed appropriately, what are the factors that may influence their inappropriate use. Factors may include cultural norms, social practices, or economic constraints. Teasing out how and why people use antibiotics in ways that can lead to antibiotic resistance will help us develop targeted interventions based on evidence rather than assumptions.


Postpartum hemorrhage is a major cause of mortality and morbidity related to childbirth in developing countries. The recommended treatment includes administration of oxytocin; however, oxytocin is a heat-labile protein, and it must be given as an intramuscular injection by skilled health care providers. To address these challenges, we developed a freeze-dried oxytocin fast-dissolving tablet (FDT) for sublingual (SL) needle-free administration. Using methods developed previously, we produced a robust FDT that maintained oxytocin stability at 40 °C, 75% relative humidity for 12 months. This formulation contains 9% sucrose, 1.5% (hydroxypropyl)methyl cellulose, 9% mannitol, 4% dextran, 1% carbomer, 1% sodium taurocholate, and 100 IU oxytocin. An in vitro study showed a > 30% reduction in tissue transepithelial electrical resistance after treatment with the oxytocin FDT, implying an increase in the permeability of the mucosal tissue to oxytocin. Anesthetized Yucatan miniature swine were administered a SL FDT, and blood was periodically collected for a pharmacokinetic study. Higher plasma concentrations were seen when larger SL doses were given. The maximum concentrations for SL and intramuscular doses in anesthetized pigs were 207 and 612 pg/mL, respectively. Whether the levels attained will be sufficient to elicit beneficial results in humans is yet to be determined. This study demonstrates the feasibility of our approach for developing a heat-stable oxytocin tablet that can be administered successfully via the SL route.The online version of this article (10.1007/s13346-017-0471-7) contains supplementary material, which is available to authorized users. Postpartum hemorrhage (PPH)—loss of ≥500 mL of blood from the genital tract within 24 h of the birth of a baby—is a major cause of mortality, morbidity, and long-term disability related to pregnancy and childbirth, with most deaths occurring in developing countries . Active Methods used to produce FDTs in our laboratory have been described previously –8. AdditWe designed 13 formulations as FDTs and performed physicochemical screening to down-select candidates for further testing; compositions of all 13 are provided in Online Resource Rapid disintegration of the FDT is essential for maximizing absorption of available oxytocin into systemic circulation, to stop postpartum bleeding as quickly as possible; it also reduces involuntary dislodging and swallowing of the tablet. Since there is limited volume of saliva in the oral cavity, FDT disintegration was tested in conditions simulating resting oral fluid volumes . We placSince oxytocin is reportedly sensitive to heat and thus requires a cold chain, one of our objectives was to develop a heat-stable form of oxytocin that could be transported and stored in low-resource areas without requiring expensive cold chain equipment. To test for oxytocin stability during long-term storage under simulated field conditions, sealed blister sheets of oxytocin FDTs in secondary foil packaging were stored in 40 °C, 75% relative humidity chambers for 12 months. Once a month, tablets were tested for oxytocin content by HPLC and for any changes in appearance such as shrinkage or collapse. The oxytocin content remained stable within acceptable limits of 90 to 110% potency for the duration of the entire study  was seen in the tissue after treatment with the oxytocin FDT, implying that this formulation may increase the permeability of the mucosal tissue to oxytocin.After oxytocin is released, post disintegration of the FDT, it is important that it be rapidly absorbed through the underlying mucosal epithelium into systemic circulation. This was achieved by incorporating permeation enhancers into our formulations; sodium taurocholate, a bile salt, was the enhancer in our lead candidate formulation. Transepithelial electrical resistance (TEER) measurements were performed on human buccal tissue samples after exposure to FDTs to determine changes in permeability of the oral mucosa upon contact with this formulation. Method details are in Online Resource Since the oxytocin FDTs are intended to be administered under the tongue, we confirmed the stability of oxytocin in saliva prior to initiation of the pharmacokinetic study. The FDTs were incubated in pooled human saliva maintained at 37 °C for an hour along with unformulated oxytocin as a control. The results from HPLC analysis indicated none to minimal proteolytic degradation of oxytocin in saliva.Sus scrofa 3 to 5 months old and weighing 15 to 20 kg were administered an oxytocin FDT under the tongue. For the control, mini pigs received 20 IU of oxytocin in a 1 mL IM injection into the right hind limb. To account for drug loss due to involuntary swallowing and dilution by excess saliva, a 20 to 30× higher dose was tested via the SL route. Although this is a high dose, levels greater than 1000 IU reportedly have been tested in women via the buccal route for induction of labor, without any adverse events [Next, a crossover pharmacokinetic study with 24-h washout was conducted to determine oxytocin plasma levels after SL administration and to compare with the standard IM route in female mini pigs, chosen due to the similarity in oral cavity architecture with humans. Ten anesthetized female, non-pregnant Yucatan miniature swine, Evaluation of SL uptake of oxytocin showed that a larger dose appeared to be associated with higher plasma levels Fig. , althougFor IM administration, the plasma levels of oxytocin obtained in unanesthetized pigs were significantly higher than anesthetized pigs (separate study). One explanation could be the effect of anesthetic agents, which can depress blood pressure. Local blood flow is a strong determinant of the rate of absorption, since it continuously maintains the concentration gradient necessary for passive diffusion to occur. It is possible that lower plasma levels seen for SL administration as well as for IM administration in anesthetized pigs can be attributed to reduced blood flow in sedated animals.Administration of drugs to the oral cavity is generally acceptable to patients, and medications can be self-administered or given by a caregiver with minimal training since no needle injection is necessary. Freeze-drying (lyophilization) is a well-established process that is traditionally used for stabilizing labile substances or processing pharmaceuticals of biological origin, such as proteins, serum, vaccines, peptides, drugs, or liposomes, which are often administered parenterally . A consiBecause of the rich blood supply and high permeability of the SL mucosa, administration via this route can result in rapid onset of action . DespiteThe feasibility study reported here demonstrates successful development of heat-stable oxytocin in a freeze-dried tablet for SL administration. While our pharmacokinetic study in pigs provides evidence for SL absorption of oxytocin into plasma, whether the oxytocin levels attained via SL administration will be sufficient to elicit beneficial results in humans is yet to be determined. The clinical potential of this presentation to prevent PPH will need to be addressed by first conducting an exploratory pharmacokinetic study in healthy non-pregnant women. Our study opens the possibility for needle-free administration of oxytocin, which would be especially useful in low-resource areas with a scarcity of skilled health workers.ESM 1(PDF 114 kb)ESM 2(PDF 100 kb)


This paper proposes the mixing design of concrete having modified sulfur-coated aggregate (MSCA) to enhance the durability of Portland cement concrete. The mechanical properties and durability of the proposed MSCA concrete were evaluated experimentally. Melting-modified sulfur was mixed with aggregate in order to coat the aggregate surface at a speed of 20 rpm for 120 s. The MSCA with modified sulfur corresponding to 5% of the cement weight did not significantly affect the flexural strength in a prism concrete beam specimen, regardless of the water-cement ratio (W/C). However, a dosage of more than 7.5% decreased the flexural strength. On the other hand, the MSCA considerably improved the resistance to the sulfuric acid and the freezing-thawing, regardless of the sulfur dosage in the MSCA. The coating modified sulfur of 5% dosage consequently led to good results for the mechanical properties and durability of MSCA concrete. Carbonation starts on the concrete surface and moves slowly to the interior of the concrete because CO2 in the air penetrates to the interior of the concrete through capillarity in the cement paste; (2) Corrosion—steel reinforcements can be destroyed by chloride ions that penetrate the concrete; (3) Freeze and thaw cycles—because concrete is porous, water is trapped and absorbed in the pores. The increased volume of water can lead to cracking when the water in concrete freezes; (4) Sulfate attack—the soluble sulfates in groundwater may react with a hydrate from cement. Wang et al. [Reinforced concrete has been widely used as construction material due to its versatility. However, several factors can cause the deterioration of concrete structures. Currently, abnormal climate and air pollution have accelerated the deterioration of concrete structures. The main factors behind the deterioration of concrete are considered to be the salt damage, carbonation, chemical erosion, frost damage, and drying shrinkage. The deterioration leads to steel corrosion and sectional-area reduction. Thus, such damage can cause the collapse of structures because it can result in strength loss and unsafe conditions. Neville explained the mechanisms of concrete deterioration : (1) Caroncrete;  Freeze afreezes;  Sulfate g et al.  recentlyg et al. ,7,8,9.Attempts to use sulfur as a construction material began around 1921 to consume the surplus sulfur from a mine named Big Doom in Texas, USA. Bacon and Davis  found thet al. [et al. [et al. [et al. [Recently, studies to improve the mechanical properties and durability of concrete by using a coating aggregate have also been conducted. Li et al.  found th [et al.  proposed [et al. . The com [et al. .To use sulfur as a construction material, it is necessary to heat the aggregate and filler as well as to melt the modified sulfur at high temperature . The hea3, respectively. Those were not much different regardless of amount of modified sulfur.The water absorptions of R40-CON, R40-NS5, R40-NS7.5, and R40-NS10 were 3.38%, 4.17%, 4.18%, and 4.20%, respectively. And the total porosities of corresponding admixtures were 7.74%, 9.48%, 9.67%, and 10.06%, severally. It was confirmed, from test results, that the MSCA increased both absorption and porosity of concrete, but the modified sulfur content at the coated aggregates slightly affected them. This result can be explained as followings: (1) the coated aggregate with modified sulfur causes its absorption to be reduced; (2) the space occupied by surplus water forms the high porosity in the MSCA concrete. The bulk densities of R40-CON, R40-NS5, R40-NS7.5, and R40-NS10 were 2.29, 2.26, 2.26, and 2.25 Mg/mThe compressive strength of the MSCA concrete decreased further when a higher amount of modified sulfur was used to coat the aggregate. There could be three reasons: (1) the remains of coated sulfur can act as impurities or light-weight aggregate in the concrete; (2) the additional quantity of superplasticizer needed to obtain an identical slump in concrete with more sulfur can lead to the formation of more pores in the cement paste; (3) the more amounts of modified sulfur owing to its low stiffness and strength result in the weaker microstructure of ITZ, leading to a decreased compressive strength.Of the specimens with 5% MSCA, the compressive strengths at 56 days in R40-NS5, R45-NS5, and R50-NS5 cylinder samples see b,c had det al. [The flexural strengths of the prism beams are plotted in et al. , which uAdditionally, the flexural strengths at 56 days of specimens with different W/C and 5% MSCA had increased when compared with each control sample, but the variation rate was slight . In other words, the 5% MSCA did not significantly affect the flexural strength loss regardless of W/C.−4, −2.10 × 10−4, −2.03 × 10−4, and −2.17 × 10−4 mm/mm, repetitively  were 0.17%, 0.16%, 0.0% and 0.14% at 30 cycles, respectively. When 300 cycles passed, the weight losses in each sample were 2.8%, 1.4%, 1.23% and 1.2%. The loss of the control specimen was almost twice as large as that of the MSCA concrete. The weight losses of the MSCA concrete with different amounts of sulfur were not significantly different.The surface of the R40-CON sample started popping out considerably from 90 cycles, and exposure of the aggregate in the concrete was seen plainly after 180 cycles had passed. On half of the surface, aggregate exposure developed after 300 cycles, as illustrated in The resistance of the MSCA concrete to sulfuric acid was investigated every 7 days for 4 weeks. The effect of MSCA on the sulfate attack resistance of concrete is shown in As compared to samples having MSCA with 5% modified sulfur, the weight losses were 1.2% in the W/C = 40%, 1.0% in the W/C = 45%, and 0.3% in the W/C = 50% samples after 4 weeks of immersion. Even though the loss of weight in two samples (R45-CON andR50-CON) with uncoated aggregate was not significantly different, the weight loss tended to be reduced with increased W/C. As test results, a higher W/C enhanced the acid resistance of the modified sulfur concrete.Corrosion probably developed on the sample surfaces in open pores that were not coated or protected by sulfur and lasted until the test ended see . The spe3 and 3340 cm2/g, respectively. OPC has an average compressive strength of 50.2 MPa at a 28-day age. Its various chemical composition and physical properties are summarized in Type I Ordinary Portland Cement (OPC) conforming to the Korean standard was used in mixture designs, and the density and specific surface area of the cement were 3.15 g/cmThe physical properties of the modified sulfur, aggregates and superplasticizer used in this experiment were summarized in 3 were fixed, the variables are summarized as follows: (1) water-cement ratios (W/C) of 40%, 45%, and 50% and (2) MSCA-cement weight ratios of 0%, 5.0%, 7.5%, and 10.0%. Note that “R” in the mixture column represents the W/C, “CON” is the control mixture with uncoated crushed aggregate, “NS” is the modified sulfur used to coat the aggregate surface, and a final Arabic numeral is the modified sulfur-cement weight ratio. The detailed concrete mixing designs are given in The target slump and air content of concrete were considered to be 150 ± 25 mm and 5.0%, respectively. While the sand-aggregate ratio (S/a) of 45% and amount of water (W) of 185 kg/mDistribution of sulfur on the surrounding aggregate was investigated by pictures taken after splitting failure, as shown in The water absorption, total porosity, and bulk density of the hardened MSCA concrete were determined after 28 days of curing in accordance with requirement of ASTM C642 . A comprThe length change of the prism specimen, as shown in 2SO4 solution at 20 ± 2 °C. The weight of the specimens before and after immersion was measured on a digital laboratory scale with a capacity of 5 kg and accuracy of 0.01 g every 7 days. The averaged data from three specimens of each mixture is used in next section of post-analysis and discussion.The sulfate attack resistance performance of the concrete was observed according to JSTM C7401 . The conThe greater the amount of modified sulfur used to coat the aggregate, the lower the compressive strength of the concrete was. This is because the modified sulfur remaining after coating can act as impurities or light-weight aggregate in the concrete. Five percent modified sulfur of MSCA barely affected the flexural strength of the concrete regardless of the water-cement ratio, while addition of more than 7.5% of modified sulfur led to quickly decreased strength.Use of MSCA in concrete slightly reduced the length change of concrete as compared with concrete not having MSCA, but the change rates did not significantly differ with variation of the melted amount of sulfur in the MSCA.Also, the MSCA considerably improved the resistance to freezing-thawing and sulfuric acid because its use resulted in high porosity and prevented a chemical reaction between acid and basic oxides. The dosage of sulfur, however, did not greatly affect the resistance.Amounts of modified sulfur exceeding 5% had bad effects on mechanical strength through a compressive and flexural test. And the modified sulfur enhanced the durability in sulfuric acid and freezing-thawing, whereas the improvement did not strikingly vary according to the amount of modified sulfur. Synthetically, in terms of improvement of durability, using 5% modified sulfur was suggested restrictively in the MSCA concrete.In this paper, the use of aggregate coated with modified sulfur was proposed as a constructional material. Based on various experiments carried out using concrete specimens made with modified sulfur coated aggregate (MSCA), the following mechanical properties and durability were observed: