Acute malnutrition to be a major health burden in the world, particularly in the developing world. Acute malnutrition is associated with more than one third of the global disease burden for children. Malnourished children are physically, emotionally and intellectually less productive and suffer more from chronic illnesses and disabilities. The nature, magnitude and determinants of acute malnutrition are determined among the general populations; however, there is a lack of evidence in the nomadic communities.p < 0.05.A cross-sectional study was conducted to assess the magnitude and factors associated with acute malnutrition among children aged 6–59 months in Hadaleala district, Afar Region. A total of 591 under-five children were included in this study, and subjects were recruited by the multistage cluster sampling technique. Data were collected by a pre-tested questionnaire and a simple anthropometric index so called mid-upper arm circumference (MUAC). The multivariable binary logistic regression analysis was used to identify factors associated with acute malnutrition on the basis of adjusted odds ratio (AOR) with 95% confidence interval (CI) and p < 0.05) and three  children in each household, unprotected drinking water sources , absence of the latrine , hand washing with soap , childhood diarrheal disease , and child vaccination .The prevalence of acute malnutrition was 11.8% . The highest prevalence (50%) of acute malnutrition occurred among children aged between 12.0–23.0 months. Childhood acute malnutrition was associated with the presence of two (AOR = 2.49, The prevalence of acute malnutrition among children aged 6-59 months was was higher than the national prevalence. The number of children in each household, drinking water sources, latrine availability, hand washing practice before food preparation and child feeding, childhood diarrheal disease, and child vaccination were identified as factors affecting the childhood acute malnutrition in the nomadic community. Protecting drinking water sources from possible contaminants, improving hand washing practices, utilization of latrine, preventing diarrheal diseases and vaccinating children integrated with the access of nutrition education is important to improve nutrition of children of the nomadic people. Under nutrition continues to be a major health burden in the world, particularly in the developing world –3. GlobaUnder nutrition among children is a critical problem. Its effects are long lasting , 8. UndeIn Ethiopia, under nutrition among children is still a common problem. Ethiopia is one of the countries with very high burden of under nutrition. In the country, under nutrition is the underlying cause of 57% of child deaths –25.Under nutrition among children depends on complex interactions of various factors, like socio-demographic –30, drin2 divided into 11 rural kebeles  with a total population of 42,845 as projected for the year 2015. It has 7516 households with an average household size of 5.7 persons per house. Under-five children account for 10.1% (4328) of the total population. As the population lives in a very scattered manner, the average population density is 14 persons/km2. Furthermore, the economy of the district is based on livestock and crop production . The likelihood of childhood acute malnutrition was 5.24 times to be higher among households who had no latrine compared with their counterparts . Children whose mothers washed their hands before food preparation and feeding with soap were less likely to be malnourished. Hand washing with soap before food preparation and child feeding can prevent childhood acute malnutrition by 79% .Childhood acute malnutrition was also statistically associated with the health status of children, like childhood diarrhea and vaccination. Childhood acute malnutrition was 2.72 times more likely to be higher among children who had diarrheal disease . This study indicated that child vaccination has a protective effect on childhood acute malnutrition. Children who ever been vaccinated were 85% less likely to be malnourished, compared with their counterparts .The prevalence of acute malnutrition among children aged 6-59 months was 11.8% . Childhood acute malnutrition was statistically associated with the number of children in each household, drinking water sources, latrine availability, hand washing practice before food preparation and child feeding, childhood diarrheal disease, and child vaccination. The prevalence of acute malnutrition reported by this study is slightly higher than the national prevalence of acute malnutrion (9%)  and findThis study showed that families who had two or above children aged 6-59 months were more likely to have childhood acute malnutrition than those who had only one child. This probably attributed to less balanced diet intake and accessibility of child healthcare decreased with more number of children per household, especially in low income families , 57, 58.In this study, it was found that acute malnutrition was associated with unprotected drinking water sources, open defecation, and poor hand washing practices of mothers. Different studies also reported that acute malnutrition was associated with drinking water sources , 31–35, This study indicated that acute malnutrition was associated with child hood diarrheal diseases. Children who had diarrheal disease were more likely to be acutely malnourished as compared with their counter parts. This finding was supported by the findings of other similar studies , 64–66. Child vaccination was also the other statistically associated variable with childhood acute malnutrition. Children who ever vaccinated were less likely to be malnourished compared with their counterparts. This finding is supported by the findings of other similar studies –47, 78. Finally, this paper determined acute malnutrition using MUAC measurement. It didn’t measure weight and height to determine global malnutrition. This paper also didn’t consider the effect of food security and access to diversified foods on childhood malnutrition. Moreover, the paper didn’t investigate demand side issues and supply side issues of systems failures with respect to poverty alleviation. The authors believed that other studies should be conducted to fill the above identified gaps.The prevalence of acute malnutrition among children aged 6-59 months was higher than the national prevalence. The number of children in each household, drinking water sources, latrine availability, hand washing practice before food preparation and child feeding, childhood diarrheal disease, and child vaccination were identified as factors affecting the childhood acute malnutrition in the nomadic community. Protecting drinking water sources from possible contaminants, improving hand washing practices, utilization of latrine, preventing diarrheal diseases and vaccinating children integrated with the access of nutrition education is important to improve nutrition of children of the nomadic people.


Aim: The aim of this work is to develop a pragmatic approach in the assessment and management strategies of patients with cirrhosis in order to optimize the outcomes in this patient population. Method: A systematic review of literature was conducted through 8 July 2017 on the PubMed Database looking for key terms, such as malnutrition, nutrition, assessment, treatment, and cirrhosis. Articles and studies looking at associations between nutrition and cirrhosis were reviewed. Results: An assessment of malnutrition should be conducted in two stages: the first, to identify patients at risk for malnutrition based on the severity of liver disease, and the second, to perform a complete multidisciplinary nutritional evaluation of these patients. Optimal management of malnutrition should focus on meeting recommended daily goals for caloric intake and inclusion of various nutrients in the diet. The nutritional goals should be pursued by encouraging and increasing oral intake or using other measures, such as oral supplementation, enteral nutrition, or parenteral nutrition. Conclusions: Although these strategies to improve nutritional support have been well established, current literature on the topic is limited in scope. Further research should be implemented to test if this enhanced approach is effective. Malnutrition has become increasingly common in end-stage liver disease . The preIn the presence of cirrhosis, malnutrition is diagnosed and defined as a deficiency in nutrients . Under nIn order to fully address the issue of malnutrition in end-stage liver disease, improvement must be made in promptly diagnosing patients in need for nutritional support.A systematic approach to screen for malnutrition in cirrhotic patients must be established to more directly address the growing problem of malnutrition. During the initial evaluation (stage 1 assessment), cirrhotic patients with immediate need for nutritional support must be identified. Subsequently, these selected patients must undergo a standardized evaluation (stage 2 assessment) leading to individualized patient care with a focus to optimize the nutritional status. This two-stage approach is summarized in The goal of a two-stage approach is to maximize the allocation of time and resources to those who need it the most to improve overall outcomes. Not every patient needs an immediate full evaluation (stage 2 assessment) for malnutrition; patients who are the most at risk should be targeted first and assessed. Malnutrition severity is strongly associated with the severity of hepatic decompensation (liver failure) in the setting of cirrhosis ,13. A ChA more thorough and comprehensive evaluation of the nutritional status can be achieved and a focused individualized approach can be pursued. This not only allows for proper apportionment of resources and time, but also provides a more targeted approach in which patients can be prioritized based on the severity of malnutrition. A study evaluating protein depletion in patients with cirrhosis concluded that the utilization of multiple nutritional assessment tools were crucial in making an accurate assessment of severity of malnutrition . End-staA standard nutritional evaluation often used in hospitals is the SGA . It is aA biochemical assessment for markers of malnutrition is commonly used for monitoring nutrition . The tesFurthermore, anthropometric testing and body mass index (BMI) are necessary tools when assessing nutritional body composition . AnthropFunctional testing is another important evaluation when assessing nutrition; this allows for a means of testing the strength and ability of the skeletal muscle . A longiThe final portion of the assessment is an evaluation of dietary intake . The besMalnutrition in the setting of cirrhosis has been noted as a reliable indicator of quality of life and key predictor of inpatient outcomes  . StudiesIn patients with cirrhosis, the recommendation for different macromolecules has changed over time and health care practitioners need to be aware of these changes so that patients are properly managed . GastroeIn general, adequate oral intake has been a difficult task for many patients facing malnutrition related to cirrhosis and other chronic ailments . PatientAlthough oral intake of food is the preferred route of nutrition, factors, such as patient weakness or inability to eat, may necessitate pursuit of alternative methods for nutrient delivery ,59. OralMalnutrition is a growing problem, especially in cirrhotic patients with end-stage liver disease. The enhancement of methods to assess and treat malnutrition is the key to optimizing patient outcomes. Assessment of malnutrition should be done in two stages, the first to identify patients at risk for malnutrition based on cirrhosis and the second to run a complete multidisciplinary nutritional evaluation on these patients. Treatments for malnutrition should make sure patients reach the recommended daily caloric and nutrient goals by increasing oral intake or by using other measures, such as oral supplementation, enteral nutrition, or parenteral nutrition. Further prospective data are needed from dedicated studies to optimize the nutritional status and outcomes in patients with cirrhosis improved through proper nutritional support of patients with advanced liver disease . Therefo


Transcriptional analysis, alkaline phosphatase activity and detection of apoptosis were used to identify transdifferentiation. Using X-ray diffraction and energy dispersive spectroscopy extracellular crystal composition was investigated. Magnesium prevented calcification in vascular smooth muscle cells. β-glycerophosphate increased expression of osteopontin but no other genes related to calcification. Alkaline phosphatase activity was stable and apoptosis was only detected after calcification independent of magnesium. Blocking of the magnesium channel TRPM7 using 2-APB did not abrogate the protective effects of magnesium. Magnesium prevented the formation of hydroxyapatite, which formed extensively during β-glycerophosphate treatment. Magnesium reduced calcium and phosphate fractions of 68% and 41% extracellular crystals, respectively, without affecting the fraction of magnesium. This study demonstrates that magnesium inhibits hydroxyapatite formation in the extracellular space, thereby preventing calcification of vascular smooth muscle cells.Magnesium has been shown to effectively prevent vascular calcification associated with chronic kidney disease. Magnesium has been hypothesized to prevent the upregulation of osteoblastic genes that potentially drives calcification. However, extracellular effects of magnesium on hydroxyapatite formation are largely neglected. This study investigated the effects of magnesium on intracellular changes associated with transdifferentiation and extracellular crystal formation. Bovine vascular smooth muscle cells were calcified using  When glomerular filtration rate decreases, phosphate (Pi) levels rise and cause severely disturbed mineral and bone metabolism affecting vascular integrity and function2. Recently, several epidemiological studies showed a significant inverse relationship between serum magnesium (Mg2+) and survival in CKD patients5.Vascular calcifications are common in chronic kidney disease (CKD) and their presence is associated with increased cardiovascular mortality, which is the primary cause of death in CKD patients2+ has been shown to effectively prevent mineralization in multiple experimental models of vascular calcification13. To date, experimental research has been focused on the role Mg2+ in the medial layer of the vessel wall, where vascular smooth muscle cells (VSMC) actively contribute to the calcification process. VSMCs transdifferentiate from contractile into osteoblast-like cells, which results in mineral deposition in the extracellular matrix, loss of contractile properties and apoptosis14. Typically, this process is characterized by the expression of genes normally restricted to bone tissue such as bone morphogenetic protein 2 (BMP2) and Runt-related transcription factor 2 (RUNX2) and increased alkaline phosphatase (ALP) activity15. Several studies suggest that Mg2+ directly prevents the upregulation of osteoblastic gene expression and thereby blocks VSMC transdifferentiation and subsequent mineralization17. However, extracellular inhibition of Mg2+ on calcium (Ca2+)-Pi particle formation, which is an important driving force for the onset of calcification, has largely been neglected18. Interestingly, it has been suggested that Ca2+-Pi nanocrystals, rather than Pi, may drive osteoblastic transdifferentation of VSMC19, illustrating the relevance of crystal inhibiting effects of Mg2+. Though poorly studied in detail in the context of VSMC calcification, the extracellular crystal inhibitory properties of Mg2+ are profound and well known in both biological and non-biological systems22.Mg2+ on both intracellular changes associated with vascular calcification  and crystal formation in the extracellular space. Therefore, in addition to mapping the effects of Mg2+ on gene expression patterns associated with osteoblastic transdifferentiation in VSMC, we studied the effects of Mg2+ on the formation of crystals in the extracellular space by scanning electron microscopy and X-ray diffraction.This study aimed to investigate the effects of Mg2. Standard culture medium contained 0.8 mM MgSO4, 1.02 mM NaH2PO4 and 1.8 mM CaCl2. VSMC were not used beyond the 16th passage and VSMC identity was confirmed by α-smooth muscle actin (α-SMA) immunofluorescent stainings. α-SMA staining was performed on cells that were cultured on coverslips (18 mm in diameter) until 80% confluent. After fixation in 4% (w/v) paraformaldehyde and blocking with 16% (v/v) normal goat serum, the cells were stained overnight (4 °C) with a mouse monoclonal primary anti-α-SMA antibody diluted  in a buffer containing phosphate buffered saline (PBS) and normal goat serum. Subsequently, cells were stained with a goat-anti mouse IgG Alexa 488 conjugated polyclonal antibody  and visualized using an Axio Imager M .Bovine aortic VSMC (bVSMC) were set up from explant culture of bovine aortic segments according to standard protocols and cultured in M199 culture medium  supplemented with 10% (v/v) fetal bovine serum , ciprofloxacine  at 37 °C in a humidified incubator with 5% (v/v) COβ-glycerophosphate . BGP requires cellular activity for its cleavage to free Pi, and was chosen in this setup to increase the medium Pi concentration to minimize Mg2+-Pi interactions prior to cellular exposure. In the high Mg2+ treatment medium, MgCl2 (Merck Millipore) was supplemented to reach a final concentration of 2 mM MgCl2. At 80% confluence, cells grown in a 12-wells plate were incubated with designated media for 14 days, which was changed every 2–3 days.For calcification, medium was supplemented with 5% (v/v) FBS and 10 mM Transient receptor potential melastatin 7 (TRPM7) was inhibited by incubation with 10 μM of 2-Aminoethyl diphenylborinate . At 80% confluence, 2-APB was supplemented in combination with the different culture media as outlined in the previous section during 14 days in a 24-wells plate.23. Briefly, a reaction mix consisting of molybdate and malachite green was added to the samples and standards and incubated for 30 minutes at room temperature. The absorbance was measured at 620 nm using a Benchmark Plus Microplate Spectrophotometer System .Pi was measured in the cell culture supernatant (or culture medium) calorimetrically using the malachite green method as described elsewhere2+ deposits present. The Ca2+ concentration in the supernatant was determined by the o-cresophthalein complexone method. o-cresophthalein color reagent (Sigma) was incubated with the samples and standards and the absorbance was measured immediately at 570 nm, as described previously24. Subsequently, the cells were neutralized in PBS and lysed in 0.1 M NaOH/0.1% (w/v) sodium dodecyl sulfate for total protein isolation. Ca2+ concentrations were normalized for total protein as determined by Pierce BCA protein detection kit according to the manufacturer’s instructions .Cells were decalcified with 0.1 M HCl for 5 minutes at room temperature with gentle rocking, which effectively dissolved all Ca2+. Cultures were washed with PBS, fixed in 4% (v/v) formalin for 15 minutes and washed with demineralized H2O before staining with 2% (w/v) aqueous Alizarin Red (Sigma) for 5 minutes.Calcification was visualized by the Alizarin Red S staining method for CaGAPDH expression levels.Total RNA was extracted from VSMC using TRIzol (Invitrogen) and treated with DNAse  to remove genomic DNA. cDNA was synthesized from 1.5 μg total RNA by Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen) for one hour at 37 °C. The primers used for PCR amplification are shown in Table p-nitrophenyl phosphate (sigma) into p-nitrophenol in a basic buffer by ALP by p-nitrophenol production. The reaction was incubated for 30 minutes at 37 °C and the absorbance for p-nitrophenol was measured at calorimetrically at 410 nm for both p-nitrophenol (Sigma) standards and samples. One unit (U) was defined as the production of 1 µmol p-nitrophenol per minute per gram protein.Cells grown in 12-well plates and cultured in designated media for 2, 8 and 14 days were lysed with 1% (v/v) Triton X-100 in PBS containing protease inhibitors. ALP activity was determined in the total lysate as the hydrolysis of Occurrence of apoptosis was measured using the Annexin V-FITC Apoptosis Detection Kit . Due to the presence of calcifications, the method for adherent cells was used according to the manufacturer’s instructions. Briefly, cells were incubated with Annexin-V-FITC and propidium iodide in binding buffer for 5 minutes at room temperature. Apoptotic cells were detected by immunofluorescence microscopy using a FITC and Texas Red filter on an Axio Imager M (Zeiss). Apoptosis was quantified using ImageJ software  by calculating the ratio of the area positive for FITC signal versus total area, as the mean of multiple captures in 3 replicates per treatment per time-point.2 were collected and purified. Supernatants were centrifuged for one hour at 16 000 × g19. The pellets containing the nanocrystals were washed with demineralized H2O and then re-centrifuged. Subsequently, the crystal pellets were dried and used for analysis. One measurement represents the crystals formed in a total of 7 wells containing 2 ml of culture medium in the BGP treated cultures. As less material was formed in the BGP cultures supplemented with Mg2+, one measurement represents the crystals formed in a total of 21 wells containing 2 ml of culture medium in order to reach sufficient amounts to detect by X-ray diffraction. For X-ray diffraction analysis, diffractograms were measured on a PANalytical Empyrean  in transmission mode with fine-focus sealed tube, focusing mirror and PIXcel3D detector, using CuKα radiation. The samples were measured in a capillary, using 0.5 mm soda glass capillaries with a wall thickness of 0.01 mm. For scanning electron microscopy (SEM)  in combination with energy-dispersive spectroscopy (EDX) for elemental analysis  the crystal pellets were transferred onto copper tape and coated with carbon. High-resolution pictures were obtained using an Everhart-Thornley SE detector. Accelerating voltage was 5 kV for morphological observations and 15 kV for micro-elemental analyses. Due to the use of BGP as calcification inducer, a cell-free control could not be included as cellular presence is necessary to cause Pi accumulation in the medium (data not shown).Cell culture supernatants of BGP-treated cells in 6-well plates with and without 2 mM MgClP < 0.05 was considered statistically significant.Parametric data were analyzed by One-Way ANOVA with Tukey’s post-hoc test to correct for multiple comparisons using PRISM software . Non-parametric data as identified by Shapiro-Wilk test for normality were analyzed using Kruskall-Wallis analysis with Dunn’s correction for multiple analysis. Time-course data was analyzed using a Two-Way ANOVA. All data are shown as mean ± SEM. 2+ supplementation led to significantly higher Pi concentrations of 7.6 ± 0.8 mM after 14 days. In contrast, BGP treatment in cell-free conditions did not lead to increased Pi concentrations under the same conditions (data not shown).BGP is a Pi-donor that requires enzymatic cleavage in order to release Pi. As Pi exposure is one of the decisive factors in the calcification process, the Pi concentration of the cell culture supernatant was assessed after 2, 8 and 14 days of treatment . Ca2+ deposition was completely prevented by 2 mM Mg2+ in our model (3.5 ± 0.3 µg/g protein Ca2+).The effect of BGP on the development of calcifications was studied using cellular Ca2+ involved in the prevention of VSMC calcification, gene expression levels of ACTA2 and osteogenic transcription factors RUNX2 and BMP2 were assessed after 2, 8 and 14 days 6(OH)2) is the most abundant type of crystal in uremic arterial calcifications and its formation has been shown to be essential for VSMC transdifferentiation and vascular calcification27. Although it has been suggested that the potential incorporation of Mg2+ in Ca-apatite crystals (PO4)6), may reduce crystal pathogenicity and increase solubility, we did not identify any whitlockite after Mg2+ treatment. Our data suggest that Mg2+ most likely prevents crystal nucleation, rather than affecting crystal content. These findings are in line with previous studies that exclusively identified hydroxyapatite, and not whitlockite, in deposits on calcifying VSMC supplemented with Mg2+ 28. Indeed, high concentrations of Mg2+ led to less hydroxyapatite deposition in a study by Louvet et al.29. Given the lack of crystals after Mg2+ supplementation, our results indicate that Mg2+ inhibits the early phases of crystal assembly in high Pi-media. Moreover, we hypothesize that the incorporation of Pi in hydroxyapatite in the BGP condition explains the lower free Pi concentration in BGP-treated compared to the Mg2+-BGP-treated culture media.Hydroxyapatite  were shown to stimulate calcification37. Interestingly, Mg2+ delays CPP maturation in uremic serum38. These findings support that Mg2+ prevents mineralization by directly inhibiting Ca-apatite crystal formation or maturation in the extracellular space.Initial Ca-Pi particle formation in response to elevated Pi-levels has shown to occur in a cell-independent manner, subsequently initiating VSMC transdifferentiation when native VSMC inhibitory capacities diminish2+ supplementation has repeatedly shown to prevent osteogenic gene expression. As a result, osteogenic gene expression has been repeatedly considered as Mg2+ target to prevent osteoblastic transdifferentiation39. In line with this hypothesis, previous research showed that Mg2+ concentrations as low as 0.8 mM reversed established calcification in human VSMC, which could be abrogated by 2-APB treatment17. These results suggest that cellular Mg2+ uptake via TRPM7 prevents VSMC calcification17. However the role of TRPM7 is controversial, as recent evidence suggests that interleukin-18 enhanced VSMC calcification through TRPM7 activation40. In our model, TRPM7 inhibition by 2-APB did not affect the Mg2+ rescue.MgACTA2 expression and α-SMA protein expression were preserved in response to BGP supplementation. Although our bVSMCs strongly calcified, BGP treatment did not result in osteogenic conversion as demonstrated by stable expression of BMP2, RUNX2 and ALPL among treatments14. Interestingly, both mRNA expression of BMP2 and ACTA2 increased over time. However, these observations were irrespective of treatment and are therefore not related to osteoblastic transdifferentiation of the bVSMC. The only transcriptional response observed during BGP-induced calcification was upregulation of the OPN gene after 14 days, which was prevented by Mg2+. Increased OPN expression is associated with calcification42. OPN is an inhibitor of calcification and potently inhibits hydroxyapatite growth and OPN upregulation has been shown to reflect a protective mechanism in response to the phosphate- and hydroxyapatite-rich environment by VSMC45. The absence of OPN upregulation in Mg2+-supplemented BGP cultures may therefore be explained by the lack of Ca-Pi formation. Moreover, OPN is only increased at 14 days after calcification was already manifested, suggesting it to be resulting from calcification rather than causing. In addition to osteoinductive signaling, apoptosis has been shown to induce the progression of calcification25. Our results indicate that calcification precedes apoptosis, as apoptosis was only detected after 14 days of BGP treatment when calcification was already manifested. In our setup, apoptosis is likely the result of exposure to Ca-Pi crystals, rather than a causative factor for calcification46.bVSMC are characterized by high basal expression levels of ALP, which makes them prone to calcification. Despite their susceptibility to calcify, the bVSMCs are contractile and do not present any signs of osteoblastic transdifferentiation, as high levels of 2+ effectively abrogates this through upregulation of calcification inhibitors and downregulation of osteogenic genes48. Indeed, we show the effective inhibition of Mg2+ in VSMC calcification. However, in contrast to previous studies, our results suggest that calcification is driven by extracellular hydroxyapatite formation independent of osteogenic transdifferentiation in bVSMCs. While many studies show the association between osteogenic transdifferentiation and vascular calcification, it remains debatable whether this transdifferentiation is an undisputable prerequisite for the development of mineralization49. Calcification represents the final common pathway of multiple pathological vascular processes50. Our results do not contradict intracellular Mg2+ effects on osteoblastic transdifferentation. However, they do highlight the presence of alternative extracellular effects on crystal formation. Overall, it is important to note that potential intracellular and extracellular pathways involved in the calcification-inhibiting capacity of Mg2+ are not mutually exclusive. Given the strong effect of Mg2+ on calcification independent of osteogenic pathways, however, the relative contribution of crystal inhibition compared to any intracellular targets may be considerable and underestimated to date.In human, rodent and bovine calcification models evidence strongly suggests that calcification is a result of VSMC undergoing osteogenic transdifferentiation and that Mg2+ on extracellular crystal growth, independent of genetic VSMC transdifferentiation. Although it has been reported previously that Mg2+ inhibits calcification, most study set-ups do not allow to discriminate between extracellular reduction of crystal formation and intracellular inhibition of osteogenic conversion17. A limitation of this study is that while we show that TRMP7 seems not to be involved in calcification, we cannot exclude that other Mg2+ channels than TRPM7 facilitate Mg2+ entry into the bVSMCs. However, it was shown previously that TRPM7 is the main Mg2+ channel in VSMCs26. Therefore, the contribution of other transporters is likely minor. In addition, we show the effectiveness of Mg2+ primarily through extracellular mechanisms involving hydroxyapatite. In contrast to other studies, the driving force of calcification in our model is not osteoblastic transdifferentiation but mainly hydroxyapatite formation and deposition. Therefore, any intracellular effects of Mg2+ involving modification of osteogenic genes such as BMP2 and RUNX2 cannot be excluded.An important strength of this study is that our model favored to study the effects of Mg2+ in preventing VSMC mineralization involving direct extracellular Ca-apatite crystal inhibition. An increasing body of studies now report that Mg2+ prevents vascular calcification by extracellular Pi binding. Mg2+ has been shown to reduce both vascular and non-vascular calcifications and to improve calcification propensity, favoring a non-cellular mechanism of action51. Therefore, Mg2+ may be considered an important and realistic approach to potentially reduce the risk for vascular calcification and subsequent cardiovascular complications in CKD patients. Clinical trials are warranted to further assess the clinical relevance of Mg2+ in relation to vascular calcifications.In conclusion, our findings demonstrate a role for MgSupplementary Figures


There is increasing interest in the definition, measurement and use of traits associated with water use and drinking behaviour, mainly because water is a finite resource and its intake is an important part of animal health and well-being. Analysis of such traits has received little attention, due in part to the lack of appropriate technology to measure drinking behaviour. We exploited novel equipment to collect water intake data in two lines of turkey . The equipment allowed continuous recording of individual visits to the water station in a group environment. Our aim was to identify drinking behaviour traits of biological relevance, to estimate their genetic parameters and their genetic relationships with performance traits, and to identify drinking behaviour strategies among individuals.Visits to the drinkers were clustered into bouts, i.e. time intervals spent in drinking-related activity. Based on this, biologically relevant traits were defined: (1) number of visits per bout, (2) water intake per bout, (3) drinking time per bout, (4) drinking rate, (5) daily bout frequency, (6) daily bout duration, (7) daily drinking time and (8) daily water intake. Heritability estimates for most drinking behaviour traits were moderate to high and the most highly heritable traits were drinking rate (0.49 and 0.50) and daily drinking time . Genetic correlations between drinking behaviour and performance traits were low except for moderate correlations between daily water intake and weight gain . High estimates of breeding values for weight gain were found across the whole range of estimated breeding values for daily water intake, daily drinking time and water intake per bout.We show for the first time that drinking behaviour traits are moderately to highly heritable. Low genetic and phenotypic correlations with performance traits suggest that current breeding goals have not and will not affect normal water drinking behaviour. Birds express a wide range of different drinking behaviour strategies, which can be suitable to a wide range of environments and production systems. There is increasing interest in defining, measuring and using traits that are associated with water use and drinking behaviour as part of genetic selection for biological efficiency in livestock. This reflects at least three issues. (i) Although water is provided ad libitum , it is nAs a research area, the measurement of drinking behaviour and the definition of associated traits have received less focus compared to feeding behaviour and its associated traits . Unlike In this study, we used the system for recording water drinking that we described in  to measuRecords of visits to electronic drinkers were collected on male turkeys from two genetic lines: (1) Line A provided 6,443,058 events from 27,415 birds aged 6 to 9 weeks and (2) Line B provided 4,680,350 events from 12,956 birds aged 10 to 13 weeks. Birds from Line A were from a paternal type line, which was selected for feed efficiency, breast meat yield and growth, whereas birds from Line B were from a maternal type line, which was selected for reproductive performance and feed efficiency. In addition, both lines were selected for leg health and fitness traits. In a previous study, we used these lines to develop a methodology for estimating drinking bout criteria in turkeys [2 or 2.5 birds/m2 at the end of rearing for the heavier Line A, in accordance with current welfare recommendations [Birds from each line were hatched every week for a period of 138 weeks, with individual hatches placed and reared in separate sheds. Drinking behaviour data were collected in pens equipped with water stations (see below). The pen was equipped with conventional group feeders hanging on feeding lines that were distributed throughout the shed, which provided ad libitum access to commercial feed, and with 16 electronic drinkers that were placed as a line on one side of the pen, which provided ad libitum access to water. The pen measured 14.8 m × 6.1 m, which corresponds to a maximum of 52 kg/mCustom-made, electronically controlled drinkers were used in the experiment; they allowed continuous recording of drinking behaviour throughout the experiment for each bird. At the start of the experiment, each bird was fitted with a passive radio-frequency identification (RFID) transponder on the leg, which recorded bird presence inside the drinker. Previous observations suggested that transponders did not affect the birds’ behaviour in any way . Access Although visits to a drinker were the recorded trait, previous work had suggested that aggregating such visits into a bout is more appropriate since only the latter is consistent with biological principles , 10. Whipdf is the probability density function for a normal mixture model, p is the proportion of intervals in the first distribution, x is natural log-transformed interval length between visits. The bout criterion was defined at the intersection point between the two distributions. This method showed that after the end of a given bout, the probability of a bird initiating the next bout is low and increases with time since the last bout, which is consistent with the principles of thirst and satiety . Each bThe calculated bout criterion was 665 s for Line A and 672 s for Line B and was used to estimate eight drinking behaviour traits: (1) number of visits per bout (VPB), (2) water intake per bout (g) (WPB), (3) drinking time per bout (s) (DTPB), (4) drinking rate at bout level (g/s) (DR), (5) daily bout frequency (DBF), (6) daily bout duration (s) (DBD), (7) daily drinking time (s) (DDT) and (8) daily water intake (g) (DWI). We estimated each of these drinking behaviour traits for each bird in both lines. Traits were analysed at three levels: per visit , per bout  and per day . All traits were defined based on their potential relevance to bird health and performance, such as time spent in proximity to drinkers and daily water intake, and with emphasis on their potential to identify different drinking behaviour strategies that birds may use to fulfil their water requirement .For performance traits, data consisted of four traits that are routinely recorded by Aviagen as part of the genetic evaluation programme Table . PerformThe first step involved estimation of genetic parameters for the drinking behaviour traits in each line separately. For the genetic analyses, all drinking behaviour traits were adjusted for body weight at the start of the test to avoid bias. Pedigree information was available for four generations and included between 100,000 and 130,000 birds per line. Combined multivariate analyses were used to estimate genetic correlations between drinking behaviour and performance traits. Phenotypic correlations between these traits were estimated only for traits that had been measured during the same test period, i.e. drinking behaviour traits and AWI or WWT.y is the vector of observations of the traits, b is the vector of fixed effects accounting for the interaction between hatch-week, pen, and contributing mating group , a is the vector of additive genetic effects, c is the vector of permanent environmental effects, and e is the vector of residuals. X, Z and W are incidence matrices that relate b, a and c to y. For birds of Line A, classes of interaction effects consisted of 123 hatch weeks, 16 individual drinking units in the water station and 46 mating groups; for birds of Line B, classes of interaction effects consisted of 89 hatch weeks, 16 individual drinking units in the water station and 40 mating groups. The interaction effect had 553 classes for Line A and 415 classes for Line B. The assumed co(variance) structure was:A and I are the additive genetic relationship matrix and identity matrix, respectively, and G, C and R represent the variance and covariance matrices of additive genetic effects, permanent environmental effects, and residual effects, respectively. All analyses were performed by restricted maximum likelihood using the VCE software [The following multi-trait variance component model was used:mentclasspt{minimaDrinking behaviour strategies were analysed with reference to WWT and AFI recorded after the water test. These analyses should help determine whether selection for either AFI or WWT is feasible, with respect to a given drinking behaviour strategy that is exhibited at the same time. To demonstrate this, estimated breeding values (EBV) were calculated for the four drinking behaviour traits DWI, DBD, DDT, and WPB, and for the two performance traits, WWT and AFI, using the PEST software . The EBVThe 6,420,769 visits for Line A and 4,666,698 visits for Line B were collapsed into 5,632,769 and 3,763,466 bouts for Lines B and A, respectively. Table Table Phenotypic correlations (Table Estimates of genetic and phenotypic correlations between drinking behaviour traits and performance traits are in Table The estimated genetic correlations between drinking behaviour traits and performance traits were low to moderate (−0.16 to 0.46 in Line A and −0.22 to 0.47 in Line B). Genetic correlations of drinking behaviour traits estimated at the visit level (VBP and DR) with performance traits were in general low, with the highest correlations between DR and WWT (0.18 in Line A and 0.27 in Line B). Similarly, genetic correlations of traits estimated at the bout level  with performance traits were in general low, apart from moderate correlations between AWI and WPB (0.29 in Line A and 0.22 in Line B), and between AWI and DBF (0.28 in Line A and 0.26 in Line B). Genetic correlations of the three drinking behaviour traits estimated at the bout level with AFI, WWT and WFT differed between the two lines. Corresponding estimates were more similar between the lines for AWI. Estimates of genetic correlations between daily time-related drinking behaviour traits  and performance traits were relatively low but differed between the two lines, except for AWI. DBD and DDT were moderately correlated with AWI, while genetic correlations between these two traits and other performance traits were generally low. Genetic correlations between drinking behaviour traits and WWT and between drinking behavior traits and WFT were in general different (when significantly different from zero).Figure This is the first report that provides estimates of the genetic basis of drinking behaviour traits and their relation to biological performance in any livestock species. The equipment used in this study recorded outputs on the basis of visits to a drinker, e.g. duration of a visit and amount of water consumed. It was suggested that such traits are of limited biological value , since tWe developed a method that clusters recorded intervals between drinking visits into drinking and non-drinking-related activity, where traits related to drinking activity intervals indicate that a bird is most likely to be in the proximity of a drinker during the bout period, but is not necessarily drinking water. It should be noted that bouts defined in this manner consisted of a small number of visits (1.14 to 1.24 visits per bout); previous studies on the feeding behaviour of poultry have suggested that a bout  may consist of a larger number of visits to the feeder , 15. AltA large sample size of birds from two lines was used for this study. Line A included birds from a paternal type line, aged 6 to 9 weeks during the experiment, while Line B included birds from a maternal type line, aged 10 to 13 weeks during the experiment. The age of the birds was different due to the experimental protocol used in the commercial settings, thus we performed no statistical comparison between the lines. Birds from both lines showed a clear separation of drinking behaviour into bouts. At the phenotypic level, we previously showed that the organisation of drinking behaviour into bouts is similar in these two lines, in spite of differences in performance goals and age . Birds fAfter having defined the biological traits associated with drinking behaviour, we estimated their heritabilities. We observed some differences in the estimates of heritability between the lines for the same trait (e.g. number of visits per bird); in general, these were lower in Line A than those in Line B, which may reflect the difference in age of the birds when tested, although there is no a priori expectation that the heritabilities for the same trait would be similar in the two lines. The relatively low estimate of the heritability for visit-based traits, such as 0.09 for VPB in Line A, justifies the view taken about the biological relevance of this trait. Traits that arise from random influence, such as a visit, are expected to have little genetic basis. All other traits showed moderate to high levels of heritability, with the highest heritability estimated for DR (0.40 in Line A and 0.50 in Line B). Drinking behaviour traits expressed on a daily basis , which may be of commercial relevance, also showed moderate heritabilities in both lines (~0.30 to 0.40).The direction and degree of the estimates of genetic correlations between drinking behaviour traits were as expected, given the biological principles that underpin drinking behaviour and water use in birds . For exaAs previously indicated, there is increasing interest in the daily water intake of livestock and its incorporation in genetic selection programmes , 16. TheEstimates of genetic correlations between drinking behaviour traits and performance traits were low in this study, apart from moderate correlations between the water-related traits with weight gains during the water and feed test periods. The latter moderate correlations were anticipated, since water intake is directly linked to food intake and hence body weight gain . The absIn addition to estimating the association between performance and drinking behaviour traits, our study also aimed at identifying the potential for selection for different drinking behaviour strategies without compromising present or future improvements in performance goals. It may be beneficial to breed birds with different drinking behaviour strategies that can perform well in different environmental conditions . In ordeThis study suggests that, if desired, drinking behaviour traits of biological significance could be incorporated into genetic selection programmes for turkeys, since they are moderately to highly heritable. Since some of these traits are correlated with each other, only a small number of drinking behaviour traits would have to be included in balanced breeding programmes. The defined drinking behaviour traits demonstrated low genetic and phenotypic correlations with performance traits, which suggest that future selection for favourable drinking behaviour would be possible without compromising other selection goals. Our findings suggest that defined drinking behaviour traits have a potential to be used in turkey breeding programmes.


Congenital pouch colon (CPC) or short colon syndrome is a rare type of anorectal malformation(ARM). Type V is the rarest form of CPC. We present a 1-day-old male child with type V CPC with prune belly syndrome and congenital anterior urethrocutaneous fistula (CAUF). A full-term male baby, third in birth order, born by vaginal delivery to a 24 year mother, was brought to our hospital with an absent anal opening and hugely distended abdomen on day 1 of life. Antenatal period was unsupervised. On examination, the abdomen was distended and flabby, with visible loops of intestine and peristaltic movement. The scrotum was empty, and testes were impalpable on both sides. The perineal examination revealed absent anal opening and well formed median raphe with a flat perineum. Phallus was flat with a large urethrocutaneous fistula on ventral side . There was no chordee and dorsal hooded prepuce. Penoscrotal transposition was present. A plain abdominal and a cross table prone lateral radiograph showed a large air fluid level covering more than half of the width of the abdomen and crossing the midline, suggestive of a high anorectal anomaly with pouch colon. An abdominal ultrasonogram showed a distended urinary bladder and bilateral grossly hydronephrotic kidneys, with bilateral dilated and tortuous ureters and distended bowel loops. Initial hematological investigation showed normal renal parameters, hemogram and electrolytes. After initial resuscitation, colostomy was planned. Exploration revealed no muscular tissue in the abdominal wall except for the fascial layer. Recto sigmoid was replaced by a pouch like structure with a thick and wide colovesical fistula. There was an abrupt transition from the normal caliber proximal bowel to the distal dilated pouch, it lacked haustrations, appendices epiploicae, and taenia coli. There was also segmental dilatation of colon proximal to the normal intervening colon . The bladder was distended, and both the ureters were dilated and tortuous. The colovesical fistula was disconnected, the pouch colon was excised, and proximal colon was brought out and end colostomy was done. Segmental dilated colon was left as such to reassess in the second stage. Postoperative recovery was uneventful. Histopathological examination of the resected colonic pouch showed normal colonic mucosa and ganglion cells. In view of the normal renal function and spontaneously emptying bladder completely without any obstruction, watchful waiting was planned, and the patient was put on prophylactic antibiotics. Baby is currently on regular follow up with a plan to do abdomino-perineal pull through of colon and repair the CAUF at later date.Type V is the rarest form of CPC comprising double pouch with short intervening segment of normal colon . But theCAUF itself is a rare anomaly with less than 50 reported cases so far . MajoritManagement of isolated CAUF is not difficult and has good outcome post surgical repair, but in complex cases where it is a part of ARM and PBS, careful planning is required. Herein, type V CPC takes priority where the distal dilated pouch is excised with closure of recto-vesical with creation of end colostomy. Proximal dilated colon may be left in-situ to be assessed in second stage where it may come to the normal caliber and may be utilized in pull through.Source of Support: NoneConflict of Interest: None


Cardiopulmonary resuscitation (CPR) is associated with a high mortality rate. Furthermore, the few survivors often have severe, persistent cerebral dysfunction. A potential cause for this unsatisfactory outcome after CPR is the combination of cardiac arrest (ischemia) and the inability to restore adequate hemodynamics during conventional CPR (reperfusion), resulting in ischemia/reperfusion injury of the whole body. Therefore we developed a concept counteracting this ischemia/reperfusion injury during the process of CPR.We present data from a patient, in whom the concept of a novel controlled automated reperfusion of the whole body (CARL) was applied after 120 min of CPR under normothermic conditions. The patient survived without cerebral deficits and showed full recovery of all organs after prolonged cardiac arrest (CA) except for the spinal cord, where a defect at the level of the 11th thoracic vertebra caused partial loss of motoric function of the legs.This is the first reported clinical application of CARL after CA. The implementation of CARL resulted in unexpected survival and recovery after prolonged normothermic CA and CPR. In synopsis with the preclinical experience in pigs this case shows, that the new concept of CARL treating ischemia/reperfusion during the CPR may be an important element within the future treatment of CA.www.germanctr.de) under No.: DRKS00005773 on July 28th, 2015.Trial was retrospectively registered in the “German Clinical Trials Register” ( Despite continuous efforts, cardiopulmonary resuscitation (CPR) is still associated with high mortality and many survivors suffer from neurological complication –3. CurreWe report the case of a 44-year-old woman who underwent CPR for 120 min after CA followed by CARL for 60 min. The patient experienced an acute onset of severe chest pain at her home that was followed by CA. Family members called emergency services and started basic life-support procedures. When the medical team arrived, the patient was not responsive to several cycles of advanced circulatory life support. In this devastating situation, the onsite team decided to transport the patient to our medical center to obtain access to a mechanical circulatory support device. During transport, the medical team continued CPR with chest compressions using the LUCAS™-system .The patient arrived at the hospital 90 min after the initial CA. Under the conditions of ongoing CPR, her arterial blood pH at this time was 6.8. Decision for CARL was made by an interdisciplinary team of a cardiologist, cardiovascular surgeon and an intensive-care physician and cannulation of the femoral artery and vein in preparation for the CARL treatment was started immediately. However, access to the very small and spastic femoral artery was difficult and time-consuming. Finally, after successful femoral cannulation  and a total time of exactly 120 min of CPR, blood circulation was provided via a new type of extracorporeal circulation device . This device was developed by our group in order to facilitate CARL, i.e. treatment of the previously ischemic whole body. Chest compressions were terminated with the start of the CIRD. In order to prevent ischemia of the leg, an additional sheath  was been inserted into the femoral artery distal of the arterial cannula and connected to the arterial cannula.The CIRD  was used to apply the novel post-cardiac arrest reperfusion protocol CARL, which has been designed to limit the extent of ischemia/reperfusion injury of the whole body (Fig. After initiation of the CIRD, the ongoing and previously uncorrectable ventricular fibrillation was converted into asystole with the application of a bolus of intravenous potassium analogue secondary cardioplegia used in cardiac surgery. Shortly thereafter, the heart regained a stable sinus rhythm. Immediately after initiation of CARL-therapy, coronary angiography was done via the contralateral femoral artery. Parallel to the CARL therapy, an acute occlusion of the proximal left anterior descending coronary artery was diagnosed. PCI was performed and a drug-eluting coronary stent was implanted. CARL was applied for 60 min, followed by continuous extracorporeal circulatory support by the CIRD for 3 days.In the ensuing days, organ function recovered stepwise. The reestablishment of satisfactory pulmonary and myocardial function allowed removal of the CIRD 3 days after CPR. The need for mechanical ventilation was prolonged due to concomitant pneumonia most likely related to aspiration during CPR. However, after 12 days, the patient was separated from the ventilator. She regained full consciousness and showed no signs of cerebral deficits. These findings were consistent with the findings in the MRI scans of the brain (Fig. Full recovery of cerebral function after 120 min of continuous CPR following CA in a normothermic setting is an extremely rare event . The couThe profound ischemia associated with CA is generally followed by severe cellular injury at the beginning of reperfusion. This triad of symptoms is known as “post-resuscitation syndrome” and has been a major challenge in the treatment of these patients , 21. TheWe are aware of the fact, that the result of this case only demonstrates one narrow example and may be not extrapolated generally to other patients. However, the above mentioned reasons are supporting the exceptional character of the medical course. Beyond that, the case report has been performed and evaluated with attention to accepted methodical elements for case studies like significance of the case, unusual general public interest, well-balanced discussion based on the relevant literature and different perspectives regarding the treatment of the medical entity .In conclusion, this is the first reported clinical application of CARL after CA. The patient suffered a severe ischemic insult after 120 min of conventional CPR following CA: She had a pH of 6.8 and a temporary spinal cord injury. The implementation of CARL using the CIRD −1.0 - system resulted in unexpected survival along with recovery from a spinal cord injury after prolonged CA and CPR. The clinical application of CARL is based on our preclinical research over many years –13. In t


Smartphone applications (“apps”) offer promise as tools to help people monitor and reduce their alcohol consumption. To date, few evaluations of alcohol reduction apps exist, with even fewer considering apps already available to the public. The aim of this study was to evaluate an existing publically available app, designed by Drinkaware, a UK-based alcohol awareness charity.We adopted a mixed-methods design, analysing routinely collected app usage data to explore user characteristics and patterns of usage. Following this, in-depth interviews were conducted with a sub-sample of app users to examine perceptions of acceptability, usability and perceived effectiveness, as well as to provide recommendations on how to improve the app.One hundred nineteen thousand seven hundred thirteen people downloaded and entered data into the app over a 13-month period. High attrition was observed after 1 week. Users who engaged with the app tended to be “high risk” drinkers and to report being motivated “to reduce drinking” at the point of first download. In those who consistently engaged with the app over time, self-reported alcohol consumption levels reduced, with most change occurring in the first week of usage. Our qualitative findings indicate satisfaction with the usability of the app, but mixed feedback was given regarding individual features. Users expressed conflicting views concerning the type of feedback and notifications that the app currently provides. A common preference was expressed for more personalised content.The Drinkaware app is a useful tool to support behaviour change in individuals who are already motivated and committed to reducing their alcohol consumption. The Drinkaware app would benefit from greater personalisation and tailoring to promote longer term use. This evaluation provides insight into the usability and acceptability of various app features and contains a number of recommendations for improving user satisfaction and the potential effectiveness of apps designed to encourage reductions in alcohol consumption.The online version of this article (doi:10.1186/s12889-017-4358-9) contains supplementary material, which is available to authorized users. Excessive alcohol consumption is a leading cause of death worldwide . There iDespite widespread availability of alcohol reduction apps on both iTunes and Google Play stores, clear evidence of their effectiveness so far appears lacking . This liThe issue of user engagement is particularly relevant to health interventions delivered via app as drop-off in usage is common (e.g. up to 95% of apps are disengaged with after 1 month) . Such loTo date, the few available evaluations that explore user preferences in relation to alcohol reduction apps tend to be limited in their generalisability, owing to the recruitment of either small or targeted sample groups (e.g. college age students) , 13, or aims to reduce alcohol-related harm by helping people make better choices about their drinking”  down”. For example, one interviewee commented that the process of recording all drinks made her feel “guilty” if she had drunk too much, while others commented that recording drinks provides a sense of “accountability”, ultimately facilitating a reduction in the amount consumed. Interestingly, self-monitoring was still considered useful even in cases where drinking had already reduced to zero units:“And I think…..even when I stopped all together, there was something still really…something to be said for still logging that, even though I was looking at a whole month and it would just be all green, and it would be like nothing, nothing, nothing, nothing, it was still quite satisfying to log that somewhere, if that makes sense….And then you’ve got somewhere to actually log that and you’ve got something that shows you the progress that you’ve made. And so that’s really, really helpful, and it’s sort of a little motivator for yourself(C10)In terms of potential effectiveness, a number of interviewees commented that the act of concurrent self-monitoring of drinking ““To an extent I was already doing it, because…I don’t know why, I just was, I thought, right, okay, you can’t drink on a school night, obviously”(C2)As highlighted in the quantitative analysis, the “no drink day” goal was most frequently set by app users. Regarding feature acceptability, interviewees favoured this goal for its simplicity, stating that it fitted easily into their pre-existing pattern of drinking as they were often already trying to incorporate drink-free days into their week. This goal was generally liked for the fact that it was clear to understand and did not involve the effort of estimating exact quantities of alcohol consumed and their associated risk:“… if I happened to have had no drinks on a day that I didn’t set as a no drink day and then it kind of racks up that way, I swear it [the app] gets confused sometimes. My goal is to not drink Monday to Thursday, but say I didn’t drink Sunday to Wednesday I think sometimes it goes, oh yes, congratulations, you’ve met your goal and I’m a bit unclear whether I have or not.”(C8)In terms of goal setting feature usability, several interviewees who set a “no drink day” goal commented that the process of pre-selecting specific “no drink days” at the start of a week wasn’t necessarily appropriate. Users expressed a wish for greater flexibility, so that goal achievements could be recognised even when an alcohol-free day did not exactly match that pre-specified. Comments also related to the fact that a week, as defined in the app, was not bounded by Monday to Sunday, but started on the day that the app was originally downloaded. This appears to have confused a number of users:“I felt that I probably couldn’t really pick a day when I’d drunk one less or I thought that would be really easy to do because I don’t drink a set amount on a certain day. I don’t like go to the pub on a certain day and think, right, I’ll have three pints today so this time I’ll just have two instead.”(C17)Fewer interviewees reported setting “drink within guidelines” or “drink on less” goals. In general, decisions regarding consumption appeared to be more clearly categorized into either consuming or not consuming, with degree of consumption not wanting to be considered by users. Interviewees mentioned that once a decision had been made to allow alcohol consumption on a specific day or in a specific setting, they were uncertain as to whether they would be able to recognize if they were drinking one less (as they had no standard benchmark against which to compare this to), or felt that as they had made the initial decision to drink, they wished to do so without having to respect consumption limits.“I only set one goal because I was very keen to kind of remain focused on one thing. I didn’t want to come and get lost in the app using it like a game. You know, I wanted to use it for one very specific thing.....I think I set it to drink probably within guidelines.”(C13)Furthermore, regarding feature usability, interviewees generally preferred to set one goal only. Interviewees suggested that using the app in this way helped them to isolate a single action that could be clearly achieved, and that setting multiple goals was confusing and potentially de-motivating.“No, it didn’t appeal - probably because I thought if I put some goals in I’m probably not going to stick to it, which probably makes me sound a bit naughty.”(C7)For interviewees who chose not to set any goals, reasons included wanting to use the app for purposes other than to change their drinking behaviour , or because they pre-empted their own failure and so did not wish to disappoint themselves in this regard:“It doesn’t really work for me…There’s not really any one place where I would go to consume alcohol.(C8)As indicated by our quantitative analysis, far fewer app users engaged with the “weak spot” geo-location feature. In terms of potential effectiveness, this feature appeared to lack relevance to users, with interviewees revealing that they tended not to associate one specific location with drinking and, if they did, this location was often also associated with many other activities (e.g. their home), so lacked identity as a trigger for alcohol consumption:“I think the weak spot is more not a physical place, the weak spot is because you suddenly think, oh god, I’ll go and have a drink.....Yes, your general circumstances are much more accurate, or much more relevant.”(B1)Other interviewees more commonly felt that they were triggered to drink at events or social gatherings, by an emotion (e.g. sadness), through feelings of stress, or they felt that their “weak spot” was more likely to be a “weak day”, such as a Friday or during the weekend, when social drinking is a cultural norm in the UK:“Yes. I did. I set up a few. I’m just wondering if it was meant to give me a notification, but I don’t recall ever getting one.”(C10)Interestingly, as identified through the quantitative analysis, even if a specific location was set up as a “weak spot”, interviewees did not commonly receive alerts when there, possibly because they had disabled this feature on their phone:quantify” their drinking habits. One interviewee commented that the “stats” enabled him to compete with himself each week to try and improve his ratings. Indeed, a re-occurring theme across several interviews was the importance of the “numbers”:“I like the numbers. I like to track stuff and have some figures behind it rather than just like, oh, I’ll go for a run today. I’ll be like, well, I’ll go for a run today but what’s my time from last time and how can I beat it? And I think that’s why this kind of app appeals to me. If I just put the drinks in and it just said you’re drinking too much but didn’t give any numbers behind it, I’d probably delete it within a few days”.(C17)Several interviewees commented on the type of notifications and feedback provided by the app. Specifically, a couple of users commented that this feedback had helped them to accurately “passive” than a previous diary app used for the same purpose, while another commented that it was the combination of self-monitoring and receiving feedback about drinking that was perceived as particularly beneficial:“I guess because you were filling out a diary it’s a bit more passive and the fact is that when you enter your figures on the Drinkaware app that first notification and ongoing notifications made you more aware of what you were doing and you are more inclined to do something about it”.(C13)In terms of acceptability of app notifications, one interviewee commented that this feedback made the app less “surprising” aspect within the app:“The most surprising thing actually was the amount of calories……the calories was something I hadn’t expected, so I think when I’ve looked back on it, that’s probably been quite useful to see.... You know, if you think about having a massive pizza that might be a 1,000 calories, I’d think twice about eating a really big pizza. But having ten pints in any night, I would link it to be unhealthy and costly and feeling a bit bad the next day, but I wouldn’t have thought before that it was also calorific.(C17)Within the app, users are provided with several forms of feedback on their drinking behaviour, one of which presents the calories consumed that week in alcohol, along with a real food item equivalent Regarding perceived effectiveness of app feedback, interviewees varied greatly in their opinions of how useful this was, with some commenting that calorie feedback is more motivating and informative than general feedback on the amount of units consumed, while others suggested that this information wasn’t specific or personal enough to be relevant:“I think putting things in terms of money. So when it works out how much you’ve saved. I think that means a lot to people and that could really hit home as well.”(C9)“The cost for me personally isn’t so much of a driver; it’s much more just about bringing down the level of drinking and about my health as opposed to what I’m spending”.(C11)In addition, the Drinkaware app also provides users with feedback on the amount of money spent on alcohol in a particular week. Again, some interviewees reported liking this feedback and finding it motivating, while several others commented that this feedback was not relevant for them:annoying” or generally disliked. In most cases, it was unclear if it was the content of a notification that was annoying or disliked, or just that these users did not wish to receive notifications in general. For example, a couple of interviewees commented that they always turn notifications off for all apps:“I probably had it switched off, I tend to switch notifications off.”(B1)Almost one third of interviewees commented that notifications from the app had been switched off. Reasons given were that notifications were considered ““I think because they were just pinging… and I was just thinking, I don’t really want to read this right now. Obviously, and I don’t know whether they do but I guess most people check their phone when something pings in and you can be with your friends and actually maybe you wouldn’t want to be saying to your friends, I’ve just got a notification from Drinkaware”.(C14)A couple of interviewees also stated that they turned off the notifications due to a concern that others would see that these were from Drinkaware, suggesting that privacy is an important user preference:Although the quantitative findings highlight a general reduction in self-reported alcohol consumption over time in engaged app users only, the uncontrolled nature of this evaluation means that no causality can be attributed . Hence, to know more about the potential impact of the app on drinking behaviour, perceptions of effectiveness were explored in qualitative interviews directly.awareness” of how much alcohol is consumed. Several interviewees talked about how the app had helped them to be more conscious of units and/or quantities consumed, even if this awareness did not directly translate into a reduction in drinking. In general, interviewees commented that the app had made them a more “mindful” drinker or more “thoughtful” about their consumption levels:“Because, just like the name of it, Drinkaware, it made me aware of my alcohol intake….rather than sat there with a calculator, which is really boring, nobody wants to do it, all you have to do is write, large glass of wine, boom boom, you’ve got all your statistics there”.(C9)“Well, I guess like all these things, I was hoping that it would change my behaviour. I think in reality it just made me more thoughtful about how much I’m drinking, which is probably a good thing”.(C17)Across interviews, a theme was frequently discussed of increased “binge drinking” specifically, and discussed how the app had helped them to realize the “low threshold” that would place a drinker within this category:“So what I’ve learnt about my drinking habits is that probably I can do 5 days a week and not drink but on a Friday or a Saturday or a Saturday and a Sunday then I’ll drink more, so I might have half a bottle of wine on a Saturday and half a bottle of wine on a Sunday, but that almost puts me into, well it puts me into a red; it puts me in towards binge drinking”.(C14)Several other interviewees referred to an increased awareness of “tool” to enable behaviour change if a user had already decided on this goal. In other words, where interviewees reported that they had already “made a decision” or had a “drive” to reduce drinking, the app may make this process easier.“I attribute a lot of it [reduction in drinking] to the app I guess because it’s a tool isn’t it? I mean obviously I wouldn’t have achieved it without the wish to do something as well personally, the drive to cut down my drinking, but I think that app is a great tool to help you achieve that”.(C11)In terms of actual behaviour change, several interviewees stated that the Drinkaware app had proven useful in regulating their alcohol intake through provision of support, enhancing commitment and providing information. This ranged from perceptions that the app had instigated behaviour change, to a view that the app was a useful “motivation” and kept them “on track”, thus assisting in self-regulation of alcohol consumption over time:“It’s very, very easy to just slip back into how you were before or to just, yeah, go on, I’ll just have a another, and things like that. And so it’s nice to have something completely external from that, that sort of just keeps you on track.(C10)A couple of other respondents commented that the app provided “reassurance” that their levels of drinking were “healthy” or “fine”, with one individual commenting that although he had not changed his drinking patterns, this was not his intention:“No, no [app has not changed drinking]….having found that I am not drinking to excess or to a bad problematic level, it’s been quite reassuring to know the amount I’m drinking is you might say fine, so I haven’t had to change anything”.(C16)Additionally, for a small number of interviewees who were classified as “low risk” at baseline, the app provided ““To be honest, in a way, I think it’s probably made things worse, because while I might have been interested in pursuing a programme, like, you know, following that and trying to do something about it, it’s just forced me to go off the app, delete it, and forget all about the idea. Do you know what I mean? So, I might even be worse off after the app, rather than better off”.(B1)One interviewee explicitly stated that the app had no effect on their alcohol consumption , while another stated that they thought the app may have inadvertently prevented engagement with an alternative drink reduction programme that might have proven more effective:“Maybe a graph of the whole 28 days, might be a really good overview.....so you can see if it’s gone down, if it’s gone up across the month, where maybe at what point in the month it goes up and down, and then, yeah, that might be quite helpful”.(C10)During interviews, factors that may have influenced app usage over time were discussed, and interviewees provided a number of recommendations to encourage longer term use and/or greater acceptability and satisfaction with the Drinkaware app. Indeed, the main criticism that emerged across interviews was the fact that the current iteration does not allow users to easily view their progress in the longer term. Being able to view changes in alcohol consumption over time was considered important, with interviewees recommending that the app include a clear visual summary of longer term trends in drinking:“It might give you a real big overview, say if you’ve been doing it for a year, it might give you a whole overview of how you’ve done in that year and how much you’ve saved, and how much…things like that. That might be quite helpful, to really look at it quite broadly.”(C10)Interviewees suggested that mapping longer term consumption patterns to specific days of the week, months or the year or to other calendar events, may also help to facilitate self-monitoring over time:“Because it’s almost like you expect it to be kind of saved on a…because I’ve got the same email address and the same everything…I just literally swapped the device. So I was quite surprised to sort of lose all that. When you’ve done well that was quite gutting.”(C3)Relating to usability, a number of interviewees also appeared dissatisfied with experiences of “losing data”, for example, following an app update or when replacing their device or switching from the Drinkaware website to the app:“Yeah, not just generically saying you shouldn’t drink because in the long-term it’s bad for you. It’s like, well, tell me something I don’t know. But if it was personalised and said, this is the long-term effect. I mean, I would even go as far as… I know some people wouldn’t like it but I’d even go as far as saying, if you drink this amount, the risk to your health is like minus 3 years off your life.”(C17)Other recommendations included the desire to receive information from the app that is more carefully personalised to current drinking habits and lifestyle. This includes risk feedback tailored to the demographic characteristics of the user:replacements” to drinking, and also include more specific information on techniques that could be employed to help cut down on the amount of alcohol consumed:“…but the education side, how do you try and get out of the yellow [indicating ‘increasing risk’]. Like what ways can you actually…how can you change your behaviour rather than just see that you’re in the yellow.”(C5)Moreover, a number of users mentioned that they wished that the app would provide suggestions for “This mixed-methods evaluation of the Drinkaware app found that the app is disproportionately downloaded by higher risk drinkers (compared to UK averages), with high attrition observed after 1 week. Users who specifically intended to reduce their drinking, or those who self-reported consuming higher levels of alcohol prior to download, were more likely to engage with the app over time (i.e. to record drinks consistently over multiple weeks) and to utilise the main BCTs contained in the app .Based on this uncontrolled quantitative analysis of routinely collected app usage data, we remain unable to conclude with any certainty that the alcohol consumption of engaged users actually reduced as a result of continued interaction with the app. Among users who completed interviews, however, and in line with existing qualitative findings in this area , a view We intended to explore how and why users engaged with specific features contained in the Drinkaware app, and to offer recommendations to improve the design of this and similar products. Generally, for those who engaged with the goal setting feature, a preference was voiced for goal options that were simple and that built on existing patterns of behaviour, with the majority of users setting one rather than multiple goals. These findings stand in support of the results of existing evaluations of app based behaviour change interventions , 25, incRelated to this, our study also indicates that more information on the link between patterns of consumption and potential health harms may need to be included in the Drinkaware app to ensure that “no drink day” goals are not being used to compensate for, or to permit, subsequent binge drinking. Indeed, as a recent randomized controlled trial of a different alcohol reduction app suggests , care neIn many cases, users indicated a desire for the app to include options to specify personal goals, while others wanted goal setting to automatically adapt to their progress. This finding fits with a growing body of literature to show that tailoring content to individual needs, and the ability to “learn” from data previously entered, are particularly sought-after features, and may even enhance effectiveness , 28. As Our analysis of usage of the “weak spot” geolocation feature does, however, emphasize the fact that any such tailoring needs to be thoughtfully designed and implemented; for example, both the quantitative and qualitative findings presented here suggest that highlighting individual physical environment-specific triggers to drinking is not necessarily seen as useful, and was generally poorly understood by users. Interestingly, this finding reflects that of Crane et al. (2015), who also found that alcohol reduction apps that contain features to enable users to identify and restructure environmental triggers to consumption were associated with lower user ratings . Other eTaken together, these results imply that conducting a full “behavioural diagnosis” prior to designing an app, whereby a comprehensive understanding of the behaviour in question is arrived at, in addition to an understanding the context in which it is enacted, is necessary to ensure effective tailoring of app content . Indeed,As with many health related apps, drop-off in usage of the Drinkaware app was large during the first month following download . From thIn users who engaged with the app for more than 1 month, the general pattern was to enter drinking data in one session, usually at the start or end of a day. This is a particularly useful finding for those who may be attempting to develop new app features, as it would suggest that these must not rely on real-time data entry . Moreover, our qualitative interviews, like others in this area , found tWe note that our quantitative analysis did show a reduction in alcohol consumption between on-boarding and subsequent weeks in users who regularly entered drinking data into the app, although this sample is very small compared to those who originally downloaded the app . This finding is also interpreted cautiously given that “typical week” consumption estimates were based on retrospective recall (i.e. at the point of on-boarding), which differs from the diary entry approach adopted thereafter. As existing research shows, individuals’ assessments of the amount that they habitually drink tend to underestimate intake . Hence, When asked for suggestions for improvements to the app, interviewees commonly mentioned the feedback that it provided. Specifically, comments centred upon a wish for the app to incorporate a visually appealing means to overview trends in consumption over time. For example, frustration was often reported if the app had “lost” data previously input (e.g. as a result of an update), suggesting that more work may need to be conducted to optimise how information is stored, shared and presented between apps and other online dashboards or interfaces .Regarding presenting longer term trends in drinking, many interviewees mentioned that this information would give them a more meaningful way to judge progress, and could be augmented by linking drinking patterns to relevant contextual information to better understand influences on consumption. To achieve this linkage, the Drinkaware app and others like it may benefit from incorporating ecological momentary assessment techniques (EMA)  . Indeed,An additional recommendation put forward by a number of interviewees was for the app to provide more information on the effect of alcohol on well-being or daily functioning, and to give a broader holistic picture of how drinking can impact individual mood patterns. While favoured by users, it remains to be determined whether provision of this type of information will be effective in prompting actual behaviour change. That this preference was expressed would, however, suggest there are likely to be significant numbers who download alcohol reduction apps not necessarily to reduce drinking directly, but who instead wish to manage a number of non-health related outcomes. Expanding initial “motivation” options present in the Drinkaware app to include improving mental health or well-being may be a good starting point to understand this need more clearly, as would allowing users to specify multiple, possibly interacting motives for downloading this or similar products .One further, consistent theme to emerge across interviews was that the current version of the Drinkaware app offers little in the way of maintenance support once drinking had successfully reduced. This is a clear limitation given that continued monitoring of consumption (including zero consumption) is documented within the literature as an effective relapse prevention technique . Future This analysis of routinely captured app usage data enabled a large sample to be analysed, with our findings complemented by the qualitative study that permitted clarification of data abnormalities and provided further information on app usage patterns. That we studied a sample of existing users, rather than individuals who were recruited into a trial, enhances the ecological validity of our results. This study presents novel insights into how real app users perceived this product, interacted with the features that it contains and explored what is sought by those who actually download alcohol reduction apps.We do, however, acknowledge a number of limitations to this study: firstly, a reliance on routinely captured app usage data meant that we were unable to draw firm conclusions regarding the effect of app usage on drinking behaviour. This is both due to the observational nature of the quantitative analysis and to the fact that a separate, validated measure of alcohol consumption was not incorporated . As such, we recommend that future studies in this area include alternative, validated measures of alcohol consumption, obtained independently of the app itself  . This maRelated to this point, we also recognise that conclusions based on analyses of routinely collected data are likely to reflect the structure of the app and app promotion activities run by Drinkaware over the course of the designated follow-up period. For example, we note that the “no drink day” goal was the first option provided to users on a vertical list see Fig. , suggestFor the qualitative study, we were unable to recruit more than two interviewees who had since disengaged with the app. Our interviews may therefore underrepresent the views of those who didn’t find the app acceptable or effective. Indeed, this investigation is limited to analysing data provided by those who continued to engage with the app over time, meaning that the results can only be generalised to longer terms users of alcohol reduction apps, and not to those who discontinue usage or who never download these products in the first place.The Drinkaware app appears to be a useful tool for raising awareness about drinking and potentially reducing consumption in individuals already committed to making such changes. The evaluation sheds light on issues surrounding usability and acceptability of the various features of the app, and provides recommendations that generally focus on user requirements for greater personalisation and tailoring. Future evaluations should seek to adopt more rigorous designs, including independent outcome measures to draw stronger conclusions about the effectiveness of apps for reducing alcohol consumption.


PLCD1) encodes an enzyme involved in energy metabolism, calcium homeostasis and intracellular movement. It is located at 3p22 in a region that is frequently deleted in multiple cancers, and the PLCD1 enzyme is a potential tumour suppressor in breast cancer that inhibits matrix metalloprotease (MMP) 7, but the detailed mechanism remains elusive. In this study, we found that PLCD1 was downregulated in breast cancers, and the gain-or-loss functional assay revealed that PLCD1 inhibited cell migration and invasion in vitro via the ERK1/2/β-catenin/MMP7 signalling pathway. Furthermore, KIF3A was identified as a downstream mediator of PLCD1, and there was an inverse correlation between the expression of PLCD1 and KIF3A. Knockdown of KIF3A expression alone suppressed cell migration and invasion, and attenuated ERK1/2/β-catenin/MMP7 signalling that was reactivated by knocking down PLCD1 in vitro. Collectively, our findings suggest that PLCD1 acts as a tumour suppressor, by KIF3A-mediated suppression of ERK1/2/β-catenin/MMP7 signalling, at least in part, in breast cancer.Phospholipase C δ1 ( Breast cancer is the leading cause of cancer-associated death in women worldwide , mainly PLCD1, FHIT, RASSF1A, and DLEC1, is frequently deleted in multiple cancers  = 0.78 (0.63−0.97), p = 0.024; Figure PLCD1 in N0  and N1-3  breast cancers was analyzed using cBioPortal for Cancer Genomics  within The Cancer Genome Atlas (TCGA) database, and the expression of PLCD1 was much higher in N0 breast cancers compared with N1-3 breast cancers (p = 0.0264)  Figure .PLCD1 was also detected in a panel breast cancer cell lines and three normal breast tissues by RT-PCR. Expression was downregulated in MDA-MB-231, MDA-MB-468, MCF-7, T47D and ZR-75-1 cells, but not in BT-549 or SK-BR-3 cells, or in three normal breast tissues , kinesin family member 3A (KIF3A) identified and linked to PLCD1 for the first time in this context   identified KIF3A, which is required for ciliogenesis and has been linked to various cancers [It is not clear how PLCD1 regulates ERK phos-phorylation. It has been reported that E-cadherin engagement leads to ERK inhibition in a PI3K/Akt-dependent pathway in differentiating intestinal epithelial cells , and in  cancers , 29. KIF cancers . However cancers . Thus, tIn conclusion, the present study provides evidence that ERK1/2/β-catenin/MMP7 signalling is involved in PLCD1-based regulation of cell migration and invasion, and KIF3A acts as a mediator in the attenuation of ERK1/2/β-catenin/MMP7 signalling. These results expand our knowledge of PLCD1 and downstream signalling in breast cancer.2. Primary breast tumour samples, matched non-cancer tissues, and normal breast tissues were obtained from the First Affiliated Hospital of Chongqing Medical University between January 2015 and October 2015. All samples underwent a histopathological diagnosis by pathologists. All patients provided written consent, and the study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.Seven breast cancer cell lines  were studied . MDA-MB-PLCD1-expressing and control plasmids have been reported previously , 10. PLCRNA extraction was performed as previously described . Brieflyβ-actin as an internal control [(−△Ct) method. Primers used are listed in Reverse transcription and semi-quantitative RT-PCR were performed using Go-Taq polymerase , and a SYBR Green PCR Master Mix kit (Invitrogen) for quantitative RT-PCR, with an Applied Biosystems 7500  and  control . Relativ6 cells/well were plated in six-well plates 1 day before scratching using a sterile tip. Migration distance was measured under phase-contrast microscopy  at the indicated time point. Moreover, 2×104 cells were seeded into transwell chambers  with or without Matrigel, and after 16–24 hours, cells on the lower side of the chamber were counted after fixing with 4% paraformaldehyde and staining with 0.5% crystal violet under phase-contrast microscopy (Leica).Cell migration was measured using wound-healing and transwell assays, and cell invasion was also evaluated by transwell assay with Matrigel  as a barrier . BrieflyCells were washed twice with cold 1×PBS, and total lysates were extracted with RIPA lysis buffer  containing protease inhibitor cocktail  with mixing by sonication. The lysate concentration was measured using a bicinchoninic acid (BCA) standard curve. Lysates (40μg) were heated at 95°C for 5 min and separated by 10–12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After transferring onto polyvinylidene fluoride (PVDF) membranes and blocking with 5% milk, membranes were incubated with primary antibodies against PLCD1 (ab134936), MMP7 , KIF3A (#8507S), ERK1/2 (#4695), pERK1/2 (#4370), β-catenin (#8480), GSK-3β (#9315), pGSK-3β (#9323), β-actin , or active-β-catenin  overnight, followed by secondary antibodies . Membranes were visualized using ECL Plus Detection Reagents .Standard streptavidin-peroxidase immunohisto-chemistry procedures were performed following the manufacturer's instructions with an UltraSensitive TM SP Kit  as previously described . PrimaryMMP7 levels in supernatants were measured using ELISA kits  following the manufacturer's instructions . Brieflyt-test in Prism 6 software , and p<0.05 was considered significant.All data were acquired at least in triplicate and are presented as the mean ± standard deviation (SD). Values were compared with controls using the two-tailed Student