The basolateral Mg2+ extrusion mechanism in the DCT is still unknown, but recent findings suggest that SLC41 proteins contribute to Mg2+ extrusion. The aim of this study was, therefore, to characterize the functional role of SLC41A3 in Mg2+ homeostasis using the Slc41a3 knockout (Slc41a3−/−) mouse. By quantitative PCR analysis it was shown that Slc41a3 is the only SLC41 isoform with enriched expression in the DCT. Interestingly, serum and urine electrolyte determinations demonstrated that Slc41a3−/− mice suffer from hypomagnesemia. The intestinal Mg2+ absorption capacity was measured using the stable 25Mg2+ isotope in mice fed a low Mg2+ diet. 25Mg2+ uptake was similar in wildtype (Slc41a3+/+) and Slc41a3−/− mice, although Slc41a3−/− animals exhibited increased intestinal mRNA expression of Mg2+ transporters Trpm6 and Slc41a1. Remarkably, some of the Slc41a3−/− mice developed severe unilateral hydronephrosis. In conclusion, SLC41A3 was established as a new factor for Mg2+ handling.Regulation of the body Mg Disturbances in this transcellular transport process result in hypomagnesemia, which is characterized by a variety of clinical symptoms including cardiac arrhythmias, muscle cramps and fatigue2+ levels2+ reabsorption in the DCT is of major importance for the treatment of patients with hypomagnesemia.The distal convoluted tubule (DCT) is essential for magnesium Mg+ homeostSolute carrier family 41 member 3 (Slc41a3) was identified as a highly regulated gene by dietary Mg2+ intake2+-enriched or Mg2+-deficient diets after which the DCT segments were isolated. Using a quantitative gene expression microarray, the Mg2+-sensitive genes in the DCT were identified. Slc41a3 was among the most significant genes with increased expression in response to a Mg2+-deficient diet.In a recent transcriptome screening study, 2+ transporters678Xenopus laevis oocytes demonstrated Mg2+ currents for SLC41A1 and SLC41A3 using physiological Mg2+ concentrations, suggesting a channel-like function52+ channel8112+-sensitive fluorescent probes showed Na+-dependent Mg2+ transport, suggesting that SLC41A1 may be the long-sought Na+/Mg2+-exchanger7SLC41A1 resulted in a nephronophthisis-like phenotype in a patient eventually requiring renal transplantationSlc41a3, Mg2+-sensitive regulation of Slc41a1 expression was not observed in the DCT transcriptome studySLC41A3 was originally described by Quamme and colleagues as part of the solute carrier family 41 of putative Mg2+ (re)absorption. For this purpose, Slc41a3 knockout mice (Slc41a3−/−) were analysed for electrolyte homeostasis, intestinal function and renal abnormalities. Furthermore, by challenging the mice with Mg2+-deficient diets, compensatory mechanisms in intestine, kidney and brain were examined in detail. Intestinal Mg2+ absorption studies were performed using the 25Mg2+ isotope to address the functional role of SLC41A3 in the intestine.The aim of the present study was, therefore, to characterize the role of SLC41A3 in renal and intestinal Mg2+ transport, a tissue expression screening for Slc41a1, Slc41a2 and Slc41a3 was performed using RT-quantitative PCR  transcript levels showed a significant 10-fold enrichment compared to total kidney mRNA. Slc41a1 and Slc41a2 expression was not enriched in DCT compared to other segments, while the latter transporter was even significantly decreased, suggesting that SLC41A3 is the most relevant SLC41 family member for Mg2+ reabsorption in the DCT.To examine the role of the SLC41 protein family in renal MgRT-qPCR, . All thr2+ homeostasis, Slc41a3−/− mice were generated. Breeding of heterozygous (Slc41a3+/−) mice resulted in a normal Mendelian inheritance pattern in the offspring. Of a total of 181 mice, 32% were genotyped Slc41a3+/+, 46% Slc41a3+/− and 22% Slc41a3−/−. Since Shirpa  screening by the Mouse Genetics Project reported abnormal locomotor coordination of Slc41a3−/− mice fed with a high fat diet (MGI: 1918949)Slc41a3+/+, Slc41a3+/− and Slc41a3−/− mice based on behavior or external phenotype. All offspring was genotyped for the insertion of the knockout cassette and presence of the wild type Slc41a3 allele  levels were not significantly altered in Slc41a3−/− mice compared to Slc41a3+/+ mice, underlining the specificity of the hypomagnesemia in these Slc41a3−/− mice  and decreased to a similar extent as the Slc41a3−/− mice on the normal Mg2+ diet (26%), they did not reach a significant difference compared to Slc41a3+/+ mice (p = 0.14). On the low Mg2+ diet, no differences in urinary Mg2+ and Ca2+ excretion or serum Ca2+ concentrations were measured among the groups  stainings absorption. To examine the compensatory mechanisms of the renal Mg2+ wasting in Slc41a3−/− mice, the expression level of renal Mg2+ transporters was analyzed using RT-qPCR  Slc41a3 is specifically expressed in the DCT and in the intestine where Mg2+ is (re)absorbed; ii) Slc41a3−/− mice suffer from hypomagnesemia and normomagnesiuria indicating a possible renal Mg2+ leak; iii) intestinal Mg2+ transporters including Trpm6 and Slc41a1 are upregulated in Slc41a3−/− mice. Additionally, this study shows that some Slc41a3−/− mice fed a Mg2+-deficient diet develop hydronephrosis.This study identified SLC41A3 as a novel player in MgSlc41a3−/− mice point to a specific renal Mg2+ leak, mimicking the phenotype of patients with renal Mg2+ wasting19202+ levels accompanied by normal urinary Mg2+ excretion. Under normal physiological circumstances, the kidney should be able to compensate for reduced blood Mg2+ levels by increasing renal Mg2+ reabsorption. However, the Slc41a3−/− mice failed to counteract their urinary Mg2+ wasting. Moreover, the urinary Mg2+ excretion was reduced in all mice fed with Mg2+-deficient diets, showing that Slc41a3−/− mice still have the ability to raise renal Mg2+ reabsorption despite inactivation of SLC41A3. However, in Slc41a3−/− fed a low Mg2+ diet for 14 days, serum Mg2+ concentrations were 26% lower than in Slc41a3+/+ littermates. Although this finding did not reach statistically significance (p = 0.14), this suggests that the Mg2+ reabsorption capacity is impaired in Slc41a3−/− mice fed the Mg2+-deficient diet. However, longer treatment may be necessary to observe a significant reduction of serum Mg2+ concentrations. Additionally, the intestinal 25Mg2+ absorption was normal in Slc41a3−/− mice, potentially because increased Trpm7 and Slc41a1 expression compensates for the loss of SLC41A3 function.The urinary electrolyte levels in the hypomagnesemic Slc41a3 was, in contrast to its close homologue Slc41a1, highly enriched in DCT. Likewise, the expression of Slc41a3, but not of Slc41a1, in DCT is highly dependent on dietary Mg2+ intake2+ channels TRPM6 and TRPM7. TRPM6 provides the specific luminal Mg2+ uptake mechanism in kidney and intestine22+ availability, EGF, insulin, ATP and estrogens222324252+ homeostasis272+ changes2+ uptake in the DCT and SLC41A1 would serve as ubiquitously expressed general Mg2+ transporter.In kidney, Xenopus laevis oocytes by voltage-clamp, showing Mg2+ currents with a Km within the physiological range for blood Mg2+ concentrations2+ currents could not be detected (unpublished data from our lab). The function of the close homologue SLC41A1 has been examined in more detail. As SLC41A3, SLC41A1 mediates Mg2+ currents in Xenopus laevis oocytes2+ transport capacity was further established in the TRPM7-deficient DT40 cell line, where SLC41A1 restored cell growth+/Mg2+-exchanger being localized at the basolateral plasma membrane72+ transporting function of SLC41A3.The molecular function of SLC41A3 in the DCT remains elusive. SLC41A3 activity has only been examined in Slc41a3 is expressed in the intestine, Slc41a3−/− mice exhibited normal intestinal Mg2+ absorption compared to their Slc41a3+/+ littermates. However, the expression of Mg2+ transporters Slc41a1 and Trpm6 was increased in duodenum of Slc41a3−/− mice, suggesting that loss of SLC41A3 function induces a compensatory mechanism to facilitate normal Mg2+ absorption. Specifically, the upregulation of Slc41a1 is of interest because the structure of SLC41A1 is largely similar to SLC41A32+ channels and essential for their function811Slc41a1 indeed compensates for defects in intestinal Mg2+ absorption in Slc41a3−/− mice, this would suggest that SLC41A1 and SLC41A3 are functionally redundant. Future studies with Slc41a1 and Slc41a3 double knockout mice should substantiate the hypothesis that SLC41A1 and SLC41A3 function cooperatively to regulate Mg2+ handling.Although Slc41a3−/− mice is a striking finding of our study. H&E stainings showed transitional epithelia including umbrella cells lining the hydronephrotic region, excluding a cystic origin of the kidney malformation3132Slc41a3−/− mice demonstrated both transitional epithelium and connective tissue lining the cavity, suggesting that an initial hydronephrosis may have ruptured resulting in a perinephric pseudocyst.The development of hydronephrosis in Slc41a3−/− mice and, therefore, the cause of the hydronephrosis could not be identified. Importantly, only a subset of the Slc41a3−/− mice developed hydronephrosis, suggesting that Slc41a3−/− mice are more sensitive to the development of hydronephrosis, but that inactivation of Slc41a3 is not causative of hydronephrosis per se. Speculatively, the dietary Mg2+ availability could have contributed to the development of the hydronephrosis. It is widely acknowledged that Mg2+ can prevent urolithiasis by reducing the formation of calcium oxalate (CaC2O4) and calcium phosphate (Ca(H2PO4)2) stones and deposits35362+ deposits in Slc41a3−/− mice on the low Mg2+ diet despite their low urinary Mg2+ excretion. H&E stainings demonstrated a slender proximal ureter containing amorphic eosinophilic material, possibly due to stasis. Although the slender proximal ureter suggests a stenosis at the ureteropelvic junction, no anatomical cause for obstruction could be identified. It could be hypothesized that a functional defect in peristalsis caused hydronephrosis in Slc41a3−/− mice, but to substantiate this further studies are required.Hydronephrosis is normally the consequence of obstruction of the ureter resulting in fluid retention in the renal calyx and pelvisSLC41A1 was recently shown to be causative for a nephronophthisis-like (NPHP-like) phenotype in an Italian familySlc41a3−/− mice. Moreover, in contrast to the NPHP-like phenotype in patients with SLC41A1 mutations, the kidney size was markedly increased in the Slc41a3−/− mice that suffered from hydronephrosis. More patients with mutations in SLC41A1 should be identified and examined for hydronephrosis to allow final conclusions on the kidney phenotype of these patients.Interestingly, a mutation of Slc41a3 mice has identified SLC41A3 as a novel player in Mg2+ (re)absorption and potentially a new factor in the formation of hydronephrosis. Consequently, SLC41A3 should be included in genetic screenings for hypomagnesemic patients.In conclusion, our study of the 2PO4, 2.5 CaCl2, 1.8 MgSO4, 5 glucose) through the heart. The kidneys were removed, minced, and digested in collagenase , 0.6 mg/ml hyaluronidase) in Krebs buffer. The digested tubules sized between 40 and 100 μm were sorted based on GFP fluorescence by COPAS . Per mouse, 4,000 eGFP-positive fluorescent DCT tubules were collected, and an additional 4,000 control tubules were sorted from the same kidney sample without selection for eGFP-positive cells.Three C57BL/6 mice were sacrificed; kidney, duodenum, ileum, jejunum, cecum, colon, brain, lung, liver, spleen, muscle, and heart tissues were collected. For the collection of DCT material, transgenic parvalbumin-eGFP mice were used as described previously Total RNA was isolated using TRIzol total RNA isolation agent  according to the manufacturer’s protocol. Obtained RNA was precipitated in ethanol, washed, and dissolved in nuclease-free ultrapure water. RNA concentrations were measured spectrophotometrically and purity was determined: in all samples, the ratio of optical density at 260 and 280 nm wavelength was >1.8. Next, 1 μg of RNA was subjected to DNase treatment  to prevent genomic DNA contamination. Subsequently, RNA was reverse-transcribed by Moloney Murine Leukemia Virus Reverse Transcriptase  according to the manufacturer’s instructions (1 h at 37 °C). Samples were then diluted 1:10 with nuclease-free ultrapure water and stored at −20 °C until further use.http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and are shown in ® Green supermix . The total volume was adjusted to 12.5 μL with diethylpyrocarbonate (DEPC)-treated deionized H2O. RT-qPCR  was carried out using a CFX96 detection system . As a negative control, the cDNA template was substituted for DEPC-treated water. Additionally, to ensure that residual genomic DNA was not being amplified, control samples, in which reverse transcriptase was omitted during cDNA synthesis, were included in the plates during measurements. All samples were normalized to the expression level of the standard mouse-specific reference gene Gapdh−ΔΔCt) and they represent the mean fold difference from the calibrator/control group.Relative mRNA expression was assessed by quantitative real-time polymerase chain reaction (RT-qPCR). Primers used for RT-qPCR were designed using the Primer-BLAST tool (w/v) agarose gel.For primer validation, standard curves with serially diluted cDNA were generated and primer concentration was optimized to ensure the efficiencies of RT-qPCR (95–105%). The construction of SYBR Green dissociation curves after completion of 40 PCR cycles revealed the presence of single amplicons for each primer pair. Amplicon size was confirmed by electrophoresis in 1.5% (Slc41a3+/–) mice of the Slc41a3tm1a(KOMP)Wtsi strain were purchased from Knock Out Mouse Project repository  and crossbred to C57Bl/6N wild-type mice. The heterozygous offspring was used to generate Slc41a3–/– mice. Littermates were housed in a temperature- and light-controlled room with standard pellet chow and deionized drinking water available ad libitum.All experimental protocols and procedures involving animals were approved by the animal ethics board of Radboud University  and were in compliance with National and European guidelines. Heterozygous male and female . The animals were housed in metabolic cages for 48hrs  prior to collect urine and feces. Subsequently, the mice were randomly divided to a group and fed with normal  and low Mg2+ diets  (n = 10 per group per genotype) for 14 days. Blood samples were taken before and after the diets via submandibular facial vein puncture. The last 48hrs of the experiment the animals were housed in the metabolic cages again to collect urine and feces. Then, animals were sacrificed, blood was collected and kidney, brain and colon tissues were sampled and frozen immediately in liquid nitrogen for further analysis.20 2+ was measured by analyzing serum 25Mg2+ as percentage of total Mg2+ Slc41a3+/+, 10 Slc41a3−/−) were fasted  overnight on wire-mesh raised floors to prevent coprophagia. At time-point 0, mice were administered a solution containing 44 mM 25Mg2+ , 125 mM NaCl, 17 mM Tris-HCl pH 7.5, 1.8 g/L fructose. Animals were administered a volume of 15 uL/g bodyweight via oral gavage. Subsequently, blood was taken at serial time-points via a small tail-cut and collected in Microvette serum tubes . After serum collection from the coagulated blood samples, sera were digested in nitric acid  for 1 hr at 70 °C followed by an overnight incubation at room temperature. Subsequently, samples were diluted in milliQ and subjected to ICP-MS analysis .Intestinal absorption of MgAfter the sampling, the tissues specimens were immediately fixed in 4% PFA (w/v) and embedded in paraffin or frozen in liquid nitrogen. 5μm sections were cut in embedding media , mounted onto Superfrost Plus slides  and stored at −20 °C. Paraffin sections were deparaffinized and rehydrated and stained with a Hematoxylin and eosin (H&E) staining. Slides were put in Hematoxillin and differentiated in tapwater. Thereafter the slides were counterstained with Eosin Y, dehydrated and mounted. Pictures were made with a Zeiss  microscope.2+ and Ca2+ concentrations were determined using a colorimetric assay kit according to the manufacturer’s protocol . Urine volume was measured to calculate 24 hours excretion.Serum and urine total Mgt-test was used .


Rec1-resilin is the first recombinant resilin-mimetic protein polymer, synthesized from exon-1 of the Drosophila melanogaster gene CG15920 that has demonstrated unusual multi-stimuli responsiveness in aqueous solution. Crosslinked hydrogels of Rec1-resilin have also displayed remarkable mechanical properties including near-perfect rubber-like elasticity. The structural basis of these extraordinary properties is not clearly understood. Here we combine a computational and experimental investigation to examine structural ensembles of Rec1-resilin in aqueous solution. The structure of Rec1-resilin in aqueous solutions is investigated experimentally using circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS). Both bench-top and synchrotron SAXS are employed to extract structural data sets of Rec1-resilin and to confirm their validity. Computational approaches have been applied to these experimental data sets in order to extract quantitative information about structural ensembles including radius of gyration, pair-distance distribution function, and the fractal dimension. The present work confirms that Rec1-resilin is an intrinsically disordered protein (IDP) that displays equilibrium structural qualities between those of a structured globular protein and a denatured protein. The ensemble optimization method (EOM) analysis reveals a single conformational population with partial compactness. This work provides new insight into the structural ensembles of Rec1-resilin in solution. Genetically engineered biomimetic proteins that display responsiveness to external stimuli are emerging as a promising class of biomaterial for a wide range of applications including controlled drug delivery, regenerative medicine, biosensors, templating agents, and hybrid nanodevices1253Drosophila melanogaster resilin by Ardell and Andersen in 2001 opened new routes to engineer resilin-mimetic polypeptides (RMPs)123141516D. melanogaster resilin (CG15920 gene product from transcript CG15920-RA) was first reported by Elvin et al.132218Native resilin is a cross-linked elastomeric extracellular matrix protein found in many arthropods. It has long been recognized for its remarkable resilience (>92%), high fatigue life (in excess of 300 million cycles), and its role in the jumping, flying and sound production mechanisms in insectsBased on limited experimental evidence, the current hypothesis is that the properties of Rec1-resilin arise from its unique molecular composition, high degree of conformational dynamics, and its ability to interact with the environment with a high level of specificity1819202122232425® There has been significant progress towards accurate and reliable prediction of protein secondary structure from the primary amino acid sequence28In order to gain greater insight into the secondary structure of Rec1-resilin, an aqueous solution of the protein was examined experimentally using CD spectroscopy. CD spectroscopy was performed over a wide range of pH. At physiological pH of 7.4, the measured far-UV CD spectra of Rec1-resilin  displaye3031Rec1-resilin has an isoelectric point (IEP) of pH 4.8, and the protonated and/or de-protonated states of tyrosine amino acid residues (pKa~10.5) in the structure have been shown to affect the photophysical properties of Rec1-resilin in different pH environmentsq) as a function of the scattering vector (q)q) is the intensity of scattering, I(0) is the intensity at zero scattering and Rg is the radius of gyration. Rg represents the overall size of the protein at equilibrium conformation and was determined /c is the forward scattering normalized against concentration, and ∆ρ is the product of scattering length density and partial specific volume of the protein. From the primary amino acid sequence of Rec1-resilin (−6 Å−2 and 0.695 cm3 g−1 respectively using the web applet MULCh (http://smb-research.smb.usyd.edu.au/NCVWeb/input.jsp). The molecular mass of Rec1-resilin from SAXS was calculated to be 28.1 kDa with an oligomerization state ratio of 0.986 . The calculated molecular mass of Rec1-resilin from SAXS data was further validated with an experimentally measured molecular mass of 28.5 kDa using matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy  hydrodynamic diameter (Dh) of 0.1% Rec1-resilin at physiological pH  to assess any residual structureThe molecular mass (MM) of Rec1-resilin was calculated from the synchrotron SAXS data for the 0.1% Rec1-resilin solution using equation 3-resilin , the scatroscopy . This isgical pH  was obseRg/hR ratio is approximately 1.4043Rg/hR ratio for 0.1% Rec1-resilin in solution at pH 7 is ~0.82. Although not quantitative, this ratio indicates the presence of residual structure in Rec1-resilin (molten globule and premolten globule)Rg values of ~47.9 and ~50.9 Å, respectively  following a power-law relationship between the polymer length and the ensemble average Rg (equation 5)N is the number of monomers in the polymer chain (310), R0 is a constant (1.33 Å) that is a function of the persistence length of the polymer, and ν is the exponential scaling factor (0.588). For a compact structure , the Rg value is predicted to be around ~19.6 ÅRg value (~43.4 Å) and the expected Rg values for unfolded and globular proteins using predictive tools, leads to an estimate that the structure of Rec1-resilin is largely unfolded but not completely disordered in nature45For a protein molecule consisting of 310 amino acid residues, the r), as a histogram of all of the inter-atomic distances (r), was calculated by inverse Fourier transform of the scattering intensity, I(q)r) is considered to be more appropriate for Rg calculations of IDPs than Guinier’s approximation; because P(r) is a model independent function and requires the entire scattering spectrum. A similar approach has been adopted by Bernado et al.Rg values of extended chains. The shape of the P(r) distribution curve with asymmetric and extended tail region  ~200 Å  program proposed by Bernado et al.Rg results in revised Rg/hR ratios from 0.90 to 0.96. These observations support the view that Rec1-resilin is more compact than if it were a chemically denatured protein and is an IDP.To determine the equilibrium molecular size and shape of uncrosslinked purified Rec1-resilin in solution, the pair-distance distribution function, P(l region  suggestsq)] vs ln(q)) )  in the iRg and Dmax  implies that less than 100% of the protein structure is free to move and supports the findings of some secondary structure by CD spectroscopy and partial compactness from Porod and Kratky analyses.The Ensemble Optimization Method (EOM)and Dmax . This und ‘pool’  is a disribution  is the oab initio reconstruction  of Rec1-resilin from SAXS data is illustrated in 512) value of 0.58, representing the goodness of fit, was obtained for GASBOR model fits to the experimental SAXS data.A typical Rg), the pair-distance distribution function, P(r), and the Porod slope, η confirm that in aqueous solution, Rec1-resilin displays equilibrium structural features between those of Gaussian chains and collapsed polymer coils. The observations support the hypothesis that Rec1-resilin is intrinsically more compact than chemically denatured proteins yet still is qualified as an IDP. The primary structural composition of Rec1-resilin is mainly dominated by 18 copies of a 15-amino acid residue repeat sequence: GGRPSDSYGAPGGGN (Ser (position 5), a tyrosine, Tyr (position 8), a Glycine, Gly (position 9), and a Proline, Pro (position 11) are conserved in all the 18 copies. It contains a very high level of Gly (34.2 mol %) and Pro (13.8%), and it lacks hydrophobic residues with long aliphatic or aromatic side chains  entropically unfavourable. On the other hand, the cyclic side chain of Pro (the only proteinogenic amino acid with a constrained phi angle) is too stiff to make a regular secondary structure and therefore intrinsically reduces the ability to form hydrogen bonds. The conformational constraint imposed by the presence of Pro residue in the peptide chain likely lowers the transition state barrier, thereby preventing secondary structure formation. Both Gly and Pro amino acid residues contribute to the propensity of the local structure to avoid folding and preclude full extension of the polypeptides535455In this study, the use of both experimental techniques and secondary structure modelling routines reveals that Rec1-resilin in solution is an IDP with robust structural conformations that are stable over a wide range of pH and X-ray intensity. The predicted structural parameters such as the radius of gyration YGAPGGGN 18. In th. In thRge chains . The amiPro-Gly) motif is often-conserved. The presence of this motif has strongly been argued to be largely responsible for mediating high-amplitude bending motions and flexibilityPro-Gly has also been confirmed in the domains of many different ELPs including ampullate spindroin 2 (MaSp2), flagelliform silk, and elastic domains of mussel byssus thread. Amyloidogenic sequences have primarily been identified below this Pro-Gly threshold53Pro residues in ELPs and their spacing on the secondary structure and reversible self-assembly characteristics using real-time imaging. It was shown that for a combined Pro-Gly threshold of >0.6 within the hydrophobic sequences, ELPs remain substantially disordered and flexible in solutionA survey of the amino acid sequences of numerous proteins possessing rubber-like elasticity  revealed that the proline-glycine  between consecutive Pro residues. Only 21% of inter-Pro spacing contains more than 8 residues indicating low content of Pro-free regions. The frequency of Pro in the peptide sequence prevents the formation of tightly packed aggregates and gives the polymer chain structural disorder, flexibility, and elasticity. It has been reported that the introduction of long Pro-free regions into the hydrophobic domains of ELPs promotes localized-sheet formation and decreases the ability to reversibly self associateGly spacing distribution , and (3) periodic use of the structure-breaking residue (Pro) in an overall hydrophilic (low hydrophobic) environment. The composition of Rec1-resilin is dominated by hydrophilic amino acid residues including both uncharged 34.2%  and charged polar 10.19% (Asp 3.94% and Arg 5.26%) hydrophilic residues  to form cross-linked Rec1-resilin hydrogelsThe mechanism for preventing protein aggregation is traditionally discussed in terms of globular tertiary structure that promotes interfaces between polar residues and water whilst shielding non-polar residues via their burial into a hydrophobic coreresidues . Consequspacings  in Rec1-et al.293057Rec1-resilin, being an IDP and displaying remarkable functionalities, challenges the traditional notion that protein function depends on a unique three-dimensional structure. In-depth experimental research on IDPs and intrinsic disordered regions (IDRs) has been limited. Peng et al.At present, there is little definitive information on the molecular basis for Rec1-resilin’s unusual multi-responsiveness in solution, and its near-perfect elasticity in the cross-linked hydrogel state. The comprehensive structural examination in this study, using a combination of in-depth computational and experimental investigations, confirms Rec1-reslin is a member of the IDP class. This work also reveals that structural features of Rec1-resilin in dilute solution inhibit protein aggregation. Kato http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html). The naturally disordered region of Rec1-resilin was predicted from the primary amino acid sequence using the PONDR®  algorithmhttp://www.disprot.org/pondr-fit.php).The secondary structure of Rec1-resilin was predicted from the primary amino acid sequence  using thD. melanogaster CG15920 gene was cloned and expressed as a water soluble protein in the bacteria Escherichia coli with a yield of ~60 mg/L of culture. The protein was then purified by a three step non-chromatographic purification method: salt precipitation (using 20% ammonium sulphate) followed by overnight dialysis at 4 °C  and heating at 80 °C for 10 min (with stirring). The denatured proteins were removed by centrifugation at 12,000 g for 15 min at 20 °C and the resulting protein solution in supernatant was freeze dried and stored for further analysis.Synthetic Rec1-resilin construct was synthesized using a cloning strategy as reported previously13® 4x LDS sample buffer. The prepared sample was equilibrated at 95 °C for 15 min and then loaded into a SDS-PAGE electrophoresis system made from NuPAGE® Novex® 4-12% Bis-Tris Protein Gel and MES SDS 1x running buffer. The protein marker was loaded into the first lane (left) and the protein into the second lane (13For molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), about 50 μg of the protein was dissolved in 7.5 μL of MilliQ water and mixed with 2.5 μL of NuPAGEond lane . An elecond lane . CompareFor molecular weight determination by MALDI-TOF-MS, pure protein (4.8 mg) was dissolved in 30% acetonitrile, 0.1% trifluoroacetic acid (TA30) to a final concentration of 3.0 mg/ml. 1.5 μl of matrix , spotted onto a polished steel target plate  and air dried. 2 μl of protein solution (1 pmol/μl) was mixed with 2 μl matrix . 0.5 μl of sample:matrix mix was spotted onto the previously created matrix spots and air dried. Mass spectra were acquired on an ultrafleXtreme MALDI-TOF mass spectrometer  operating in linear positive mode. Instrument settings were set in flexControl software . Measurement range was set to m/z 5000 – 50,000. 5000 shots were collected for the external calibration and 20,000 shots for sample measurement. External calibration was performed using a mix of protein calibration standard I and II . Laser intensity and detector gain were manually adjusted for optimal resolution. The MS spectra obtained  were anahttp://dichroweb.cryst.bbk.ac.uk/html/process.shtml) was used to extract secondary structure information from CD spectra. Three different fitting algorithms namely CONTINFor secondary structure analysis of Rec1-resilin using a CD spectrometer , a protein concentration of 100 μg/ml was used unless otherwise indicated. The desired amount of protein was dissolved in 5 mM phosphate buffer saline  for pH 7.4 analysis. For pH 2, 4.8 and 12 the desired amount of protein was dissolved in pH corrected (adjusted using 1 M NaOH and 1 M HCl) PBS solutions. An UV quartz cuvette with path length of 1 mm was used for all the measurements at a fixed temperature of 25 ± 0.1°C. In all the cases the detector voltage remained below 600 mV and the respective buffer background was subtracted. The online fitting program interface Dichroweb  was used for low protein concentrations such as 0.013, 0.025, 0.05 and 0.1% w/v. The synchrotron SAXS resolution (extended q range) proves advantageous over bench-top SAXS in investigating structures of proteins in low concentrations, where there is no need for removing the effect of beam profile from the data (desmearing) as required in bench-top SAXS. In both beam lines, the samples were placed in a quartz capillary with temperature controlled at 25 ± 0.1 °C. In all the cases the buffer background was subtracted from the sample. Initially, the samples were carefully checked for any protein structural damage by X-rays (q regionq regionq regionr)q region using the PRIMUS programThe equilibrium structure and morphology of Rec1-resilin in Milli-Q Gradient A10 purified water was investigated using SAXS. Both bench-top and synchrotron SAXS beam lines were employed for in-depth understanding of the intrinsic structure. A bench-top NanoSTAR II SAXS (Bruker) with a rotating anode Cu Kα radiation source 1.541 Å) and a 2D detector was used to analyse protein concentrations of 0.1, 0.5 and 1% w/v. A scattering vector, y X-rays ; and wity X-rays , the sca Å and a N independent models based upon sequence and structural information was first generated. No rigid body was used in the input, and the complete random configurations of the α-carbon trace were created based upon the sequenceWith Rec1-resilin recognized to be intrinsically unfolded, the Ensemble Optimization Method (EOM) was used to fit the averaged theoretical scattering intensity from an ensemble of conformations into the experimental SAXS dataHow to cite this article: Balu, R. et al. Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin. Sci. Rep.5, 10896; doi: 10.1038/srep10896 (2015).


In patients who undergo median sternotomy to treat congenital heart diseases, a thymectomy is performed to yield better access to the cardiac system. In this study we have used MRI to evaluate the changes in size, shape and location of the thymus after midsternatomy. This case-control study was performed during 2011-2012 in Shiraz, Iran. Eligible participants between 5-17 years of age were divided into case and control groups (n=13 per group). Each participant underwent a median sternotomy at least one year prior to study entry. Participants were initially examined by a cardiologist and then referred for MRI. A radiologist examined all MRI images. The thymus was observed in all control group patients and in only 7 (53.8%) patients in the case group. There was a significant relationship noted in terms of mean age in the group whose thymus was visible and the group in which the thymus was not visible. We have observed no significant difference in thymic visibility between these two groups based on the mean age at midsternatomy. In pediatric patients undergoing cardiac surgery the possibility of remaining or regenerated thymic tissues may be evaluated by MRI. The remaining portion of the thymus may have any shape, size or location. Therefore, it can be misinterpreted as a mass if a patient’s previous surgical history and age at the time of surgery are not taken into consideration. Anatomically, this organ is situated at the anterior superior mediastinum, exactly behind the sternum and in front of large blood vessels.2 Complete correction of TOF is performed under cardiovascular bypass surgery. Long-term evidence regarding the prognosis of such patients is not available; however, most affected children are treated or at least become asymptomatic throughout childhood or at the beginning of adulthood.3The most common congenital cyanotic heart disease in adults is the tetralogy of Fallot (TOF).4,5Access to the heart and large vessels is difficult in infancy and the first months of life because of the size of the thymus. Therefore in children with congenital heart disease, a thymectomy is performed in order to facilitate cardiac surgery. This procedure leads to ectopic thymus tissue and ultimately thymus hyperplasia in the mediastinum.6,7Magnetic resonance imaging (MRI) is a suitable method for evaluating a normal or hyperplasic thymus and related tumors. When compared with computed tomography, it is safer because patients are not exposed to a higher dose of radiation.In patients who undergo median sternotomy because of congenital heart disease, thymectomy is performed to enable better access to the cardiac system. However, the main question is whether the thymus is able to regenerate after surgery. Therefore, considering the increasing prevalence of congenital heart diseases worldwide (including Iran) and few existing studies in this regard, it is necessary to evaluate the changes in the thymus after surgery and during follow-up to take the necessary therapeutic approaches. We aimed to evaluate the changes in size, shape, and location of the thymus after midsternatomy using MRI.This case-control study was performed during 2011-2012 in the MRI Center of Shahid Faghihi Hospital, affiliated with Shiraz University of Medical Sciences, Shiraz, Iran. Participants were selected according to the simple sample selection method. Eligible individuals were divided into case and control groups. The control group (n=13) consisted of individuals with no history of chest surgery or known illness who referred for MRI for any other reasons. The case group (n=13) consisted of patients with TOF who were 5-17 years of age and had undergone median sternotomy only once (complete correction) at least one year prior to the study. The one-year period was considered necessary in order to bypass any transient thymic hyperplasia that might occur in the first few months after surgery. The sample size was calculated using the simple calculation method. We excluded patients with any accompanying pathology such as DiGeorge syndrome, those who used steroids, or those who had recent infections over the previous two weeks.After obtaining written informed consent and approval from the Ethics Committee at Shiraz University, patients were interviewed to complete the related questionnaire. The other required data were collected through a review of patients' surgery reports. The included patients were subsequently examined by a cardiologist and referred for MRI. In order to study the effect of factors such as age and elapsed time from surgery we subdivided the participants into two groups - those in whom the thymus was visible through MRI and those in whom the thymus was not visible.2 turbo, and axial in and out phase. We initially aimed to obtain sagittal in and out phase images. However, considering the longer imaging time and the lack of patients’ cooperation to control their respirations, we changed to the mentioned sequences.Imaging was done using a Siemens device  with a magnetic field of 1.5 Tesla. The protocol for the obtained sequences performed by a single technician under the supervision of a radiology resident was as follows: axial HASTE sequence, axial TAll images were saved in the picture archiving and communication system (PACS) and evaluations were performed on a work station. All images and sequences were accurately examined by a single radiologist. The visibility, shape , tissue heterogeneity and homogeneity, size , and place of the organ as well as ectopic or hyperplasic tissue were accurately examined. All images were carefully examined for any random findings. Data were analyzed using SPSS software, version 15. Mann-Whitney U and Fisher’s exact tests were used as appropriate. A P value<0.05 was considered statistically significant.In the case group, there were 6 girls and 7 boys . The control group consisted of 6 boys and 7 girls . The patients’ ages ranged from 1-14 years at the time they underwent median sternotomy. The elapsed time after surgery varied from 2-7 years. The thymus was easily observed in all participants in the control group on axial HASTE images compared with only 7 (53.8%) patients in the case group (P=0.015). We found that gender did not have a significant effect on visualization of the thymus (P=0.695). The mean±SD time elapsed from surgery in those whose thymus was visible through imaging was 3.14±1.77 years and in those whose thymus was not visible, it was 3±0.894 years . For re-evaluation we divided the patients into two groups based on the time elapsed from surgery (2 years and over 2 years). There was no significant relationship between these two groups .There was a significant relationship in terms of mean age between the group in which the thymus was visible (9.7±4.23 years) compared to the group in which the thymus was not visible . The age range of the latter group was 5-9 years (median: 7 years) compared with 5-17 years (median: 10 years) in the group in which the thymus was visible. There was no significant difference regarding the visibility of the thymus based on the mean age at which the patient underwent surgery in the non-visible thymus group (4±2 years) versus the group with a visible thymus (6.43±4.23 years).None of the patients in the case group had a normal size thymus, nor was the size close to normal. 8 In our case group the thymus was visible in 53.8% of the patients by axial HASTE image. This finding suggested that more than half of the children had either a persistent or regenerated thymus after open cardiac surgery which could be attributed to the type of patient selection. We have selected patients with a higher age (over 5 years) or those who had underwent surgery only once by a single surgeon using a similar method. As the thymus consists of a wide variety of shapes and sizes, prediction of the shape and size of any remaining thymic tissues can be difficult. These three factors of a higher age over 5 years, only one surgery, and similar surgical method can enhance the visualization of the remaining portion of the thymus after mid-sternatomy, however in the superior mediastinum this leads to increased thymic identification in comparison with a report by MacDonald and Mackenzic who have reported 29% thymus identification.8In our study, the thymus was seen in all patients in the control group. In the only previous study in this regard, the thymus was seen in 92% of the patients in control group and in the remaining patients the thymus was not visible for unknown reasons.A unique feature of our study was that we chose only patients with TOF rather than different types of ongenital heart disease (CHD). Patients were operated on by a single surgeon using a similar method. All images were obtained by the same device with a standard protocol and by a single technician. Images were examined under the supervision of a radiology resident and the undesirable images were repeated until an acceptable image was obtained. Less than half of the children in our study, regardless of their age at sternotomy, did not have an identifiable thymus according to MRI after surgery. In our study the control group consisted of individuals with no history of chest surgery or known illness which referred to the MRI center for other reasons.In most patients in the case group the thymus was clearly smaller. The signal was heterogeneous in 3 patients and the shape of the thymus was irregular in 5 patients. These were normal changes after surgery, thus the remainder of the thymus could be of any shape and dimension and located in any part of the mediastinum.8 At younger ages the gland’s tissue is situated mostly in the lateral sections and therefore less tissue is needed to be removed in order to reach the heart. However, we have found no relationship between age at which surgery is performed and subsequent visualization of the thymus. The method of thymus removal is not usually mentioned in patients’ records. This issue remains controversial and it is necessary to conduct additional related studies.An important question which arises is why the thymus is seen in some patients after median sternotomy. The best explanation can be the use of different techniques during surgery. A study has reported a weak, although not statistically significant relation between the performance of cardiac surgery at a younger age and the presence of a visible thymus on subsequent imaging studies.We found a significant relationship between patient’s age at the time of MRI and visibility of the thymus, which was not found in the previous study. The mean age of the patients in whom the thymus was visible was higher. This finding could be attributed to the age of the selected patients because in younger patients the surgeon should remove more thymic tissues compared to older patients. Considering the larger size of the thymus in adolescence, after surgery the thymic residue is larger and the peripheral part of the thymus could remain in place even after removal of the thymus compared with younger patients whose entire thymus gland is removed.9,10 However, the long-term effects of reduction in the active tissue of the thymus and T lymphocytes on children who undergo related surgeries has yet to be evaluated. It is widely known that children who lack a thymus gland during the fetal period (DiGeorge syndrome) experience complete deficiency and dysfunction in T lymphocytes and have severe cellular immune deficiency. In patients who undergo sternotomy or thymectomy, it is possible that T lymphocytes may mature outside the thymus and the remaining tissue may suffice. However, the long-term effects of this defect under certain conditions such as infections have not been proven. Therefore, designing a long-term study to assess different groups of patients who have undergone median sternotomy can be beneficial.Several studies have shown that patients who undergo sternotomy and thymectomy experience long-term reduction in the amount of T lymphocytes and a disruption in their function.A limitation of the current study was the inability to evaluate the T-cell counts for our patients. However none of the patients in the case group had any important health problems or developed malignant diseases. The long-term risk of reduced/absent thymic tissue in this population with respect to infectious or malignant diseases should be evaluated. We recommend that prospective studies be conducted.In pediatric patients who undergo cardiac surgery the possibility of remained or regenerated thymic tissues should be evaluated using MRI. The remaining portion of the thymus could have any shape, size, or location. Therefore, it could be misinterpreted as a mass if the history of previous surgeries and patient's age at the time of surgery has not been considered.


There are limited longitudinal data on the associations between different social contexts of alcohol use and risky adolescent drinking.Australian prospective longitudinal cohort of 1943 adolescents with 6 assessment waves at ages 14–17 years. Drinkers were asked where and how frequently they drank. Contexts were: at home with family, at home alone, at a party with friends, in a park/car, or at a bar/nightclub. The outcomes were prevalence and incidence of risky drinking  on a day, past week) and very risky drinking  in early (waves 1–2) and late (waves 3–6) adolescence.Forty-four percent (95 % CI: 41-46 %) reported past-week risky drinking on at least one wave during adolescence (waves 1–6). Drinking at a party was the most common repeated drinking context in early adolescence ; 15 % reported drinking repeatedly (3+ times) with their family in early adolescence (95 % CI: 14-17 %). For all contexts (including drinking with family), drinking 3+ times in a given context was associated with increased the risk of risky drinking in later adolescence. These effects remained apparent after adjustment for potential confounders . Similar patterns were observed for very risky drinking.Our results suggest that consumption with family does not protect against risky drinking. Furthermore, parents who wish to minimise high risk drinking by their adolescent children might also limit their children’s opportunities to consume alcohol in unsupervised settings.The online version of this article (doi:10.1186/s12889-015-2443-5) contains supplementary material, which is available to authorized users. Considerable research has examined the place of alcohol in different societies and cultures . There hincrease risks of harmful use [Yet the evidence on whether the social context of drinking, including consumption with family, affects adolescents’ likelihood of engaging in risky drinking is unclear. The few studies of associations between adolescent consumption of alcohol with parents, and patterns of alcohol consumption in adolescents, provide conflicting evidence , 3. Two mful use ; and thamful use . HoweverThere has been limited examination of other drinking settings, particularly ones with less parental supervision, such as parties. It may well be that consuming alcohol in less supervised settings than the family home increases the risks for heavier alcohol use. Adolescents who drink with their parents may drink more either because they are already drinking and hence more likely to drink in a range of different contexts, or because drinking at home increases risky drinking. As a consequence, it is unclear what advice we should give parents about when and how to introduce their children to alcohol . Some haThe prevalence and incidence of adolescent risky drinking  in a session) and very risky drinking ;The frequency of drinking in different contexts reported in early adolescence, including parental supply and supervision;Predictive associations between the contexts in which alcohol is used in early adolescence and incident risky drinking and very risky drinking in later adolescence.This paper examines predictive associations between the contexts in which alcohol is consumed in early adolescence and risky   declined continued participation, leaving 44 study schools.A two-stage cluster sampling procedure, first by school and then by class, was used to select the study population with class recruitment at two entry points. At stage one, 45 schools were randomly chosen from a stratified frame of government, Catholic and independent private schools with the probability of selection in each stratum proportional to the number of students of that age in each school. At stage two of the sampling procedure there were two entry points, with one intact class selected from each participating school in wave 1, and a second class selected six months later (wave 2). These two classes from each participating school then formed the basis of the cohort which was followed up during adolescence a further 4 times at six monthly intervals (waves 3 to 6) and during young adulthood 3 times (waves 7 to 9) , with adult wave data (waves 7 and 8) only used to identify parental divorce and separation occurring during adolescence that was reported retrospectively by study participants in adulthood. From a total sample of 2032 invited participants, 1943 (96 %) participated at least once during the six adolescent waves. Seventy six invited participants were either refused consent by their parents or were never available for interview . The drinking contexts were: at home with family, at home alone, at a party with friends, at a pub or place to eat, in a park or on the street or at the beach, in a car, or at a nightclub.In Australia, the legal age for purchase of alcohol is 18 years. Consumption of alcohol by those under 18 years is illegal, unless under the direct supervision of a parent or another adult acting in Within each wave of data collection and for each drinking context, we identified adolescents who (a) never drank in that setting, and for those who did so, we categorised frequency of drinking in that context as occurring (b) 1–2 times for those reporting “once or twice” or (c) 3+ times for those reporting “less than monthly” or “more than monthly”. We then combined, firstly, “in a park or on the street or at the beach” with “in a car” into a single measure termed “in a park/car”, and, secondly, “at a pub or place to eat” with “at a nightclub” into a single measure “in a bar/club”. For the combined categories, drinking was categorised as 3+ times if adolescents reported drinking “less than monthly” in both contexts or “more than monthly” in at least one context. In early adolescence (waves 1 & 2), for each context we identified those adolescents who were drinking 3+ times at one or both waves, and termed this “repeated” use in that context in early adolescence; and identified those with non-repeated use as non-drinkers or those who were drinking less than 3 times in that context at both waves.Participants who reported that they drank alcohol in the week prior to interview were asked to complete a beverage- and quantity-specific one-week alcohol diary (for more details see ). We calWe identified participants using tobacco daily.We identified participants using cannabis ≥ weekly.Antisocial behaviour was assessed using 10 items from the Moffitt and Silva self-report early delinquency scale [never, once, or more than once in the last 6 months. At each wave, antisocial behaviour was identified in participants who reported multiple behaviours at least once or one behaviour more than once.cy scale , assessiMental health was assessed using the Clinical Interview Schedule (CIS-R) [ (CIS-R) , which aRural and urban areas were defined using the location of the school at study inception.Frequency of parental drinking and smoking was assessed in the course of the study. Regular parental smoking and drinking was defined as at least one parent engaging in these behaviours on most or every day.Parental divorce or separation in adolescence (by wave 6) was identified in the course of the adolescent surveys or from responses to enquiry about parental marital status in later waves if the adolescent was absent at wave 6.Prevalence and incidence of risky drinking and very risky drinking were estimated overall and separately for males and females at each wave within a multiple imputation framework (see below). The frequency of drinking in each of the drinking contexts was estimated for males and females in early adolescence (waves 1 & 2). The association between drinking with family at home and drinking in each other context in early adolescence was estimated using odds ratios.other drinking context  was estimated separately and then jointly to assess the effect of each drinking context on incident risky drinking. To estimate the effect of joint drinking locations, we generated new four level variables for each other context: not drinking in other location and not drinking at home with family (baseline category); not drinking in other location and drinking with family; drinking in other location and not drinking with family; drinking in other location and with family.The association between adolescent drinking contexts measured at waves 1 and 2 and incident risky drinking in late adolescence (waves 3–6) was assessed using repeated measures discrete time proportional hazards models . AdjustmIn these models, wave of observation was entered as three dummy variables with wave 3 as reference category in order to avoid constraining the effect of wave/time to be linear. Interactions between sex and drinking context, and sex and wave were assessed in the fully adjusted separate models. There was no evidence of interactions between any of these variables so they were not retained in the final models. All main effects and interactions were tested for statistical significance using Wald tests . All data analysis was conducted using Stata 13 .Multiple imputation  was usedAfter imputation, any transformed variables were converted back to their original scale and categorised for analysis, with adaptive rounding for binary measures . Simple Fifty one percent of the sample was female, 26 % attended a rural school at study inception, 39 % reported that at least one parent smoked cigarettes on most days, 32 % reported that their parent drank alcohol on most days, and 23 % reported that their parents were separated or divorced by wave 6.Overall, 44 % (95 % CI: 41-46 %) of participants reported risky drinking in the past week on at least one wave during adolescence. This was more common in males  than females . The frequency of risky drinking in the past week increased steadily across adolescence: from 7 % in wave 1 to 23 % in wave 6  of adolescents reported drinking repeatedly (three or more times in the past six months) in at least one context. Slightly more males  than females  reported drinking 3+ times in the past 6 months in one or more contexts in early adolescence. Figure During early adolescence, males and females who reported repeated drinking at home with family were more likely to report repeated drinking in other contexts than those who did not drink repeatedly at home with their parents Fig. . Males wTable other context, with incident risky drinking in late adolescence. In each case, adolescents who drank 3+ times in either or both contexts were more likely to report risky drinking in later adolescence than those who drank in neither context. Drinking with family also appeared to increase the risk of incident risky drinking for those drinking in each of the other contexts compared to those just drinking with family. These effects remained after adjusting for other factors known to influence risky drinking. The same pattern was found for incident very risky drinking  than among those who had done so less frequently, or not at all. Incident risky drinking was also even more likely among young people who reported drinking in contexts where they had limited (or no) parental or adult supervision .To date, the risks of varied social contexts of alcohol use have not been examined in a prospective study, while taking account of confounding. Our results challenge the rationale for parents supervising alcohol consumption by adolescents as a strategy to reduce harmful drinking. Drinking in less supervised contexts also increased risks for later risky drinking.any context, including with parents, heightens risks for later risky patterns of alcohol use. This would be consistent with evidence that earlier onset alcohol use is an important predictor of later problematic alcohol use [It is difficult, based on observational data, to infer that the associations we observed are causal. For example, the consumption of alcohol with family may be hypothesised to reflect permissive parental attitudes towards drinking . These aohol use  and thatohol use .outside the home, which additionally increased risks of “problem” drinking [Such a possibility is consistent with a Dutch cohort study of adolescents, where it was found that drinking in the home was not protective against later “problem” drinking , and spedrinking . This podrinking .This study has the strengths of prospective design and multiple waves of measurement but we did not ascertain the quantity of alcohol consumed in each of the social contexts. Further, our data may not have captured the full extent of risky drinking in this cohort because not all risky alcohol use will have occurred within the one week reference period. Both of these limitations mean that our estimated levels of risky drinking probably underestimate the total amount of risky drinking at each wave, and over time. All data were also based on self-report, but there is reasonable evidence that young people’s estimation of their alcohol use are reasonably reliable and valid when these reports are made in a confidential manner without any consequences for disclosing use, as was the case here , 31.These findings may not apply in all cultural settings . In MediSimilarly, it is also unclear to what extent these findings might apply to countries with zero-tolerance policies towards drinking in adolescence (e.g. USA). However, recent work in the United States has suggested that, as we found in this study, the most common social contexts of alcohol use among adolescents were at a party or at home, and most commonly with one or two other people , suggestA significant proportion of the Australian adolescents we studied engaged in risky drinking during adolescence, and they drank alcohol in a variety of contexts. It does not seem that early adolescent drinking with the family was protective against later risky drinking. Parents wishing to reduce their children’s alcohol use may be better advised to limit the opportunities for adolescents to drink alcohol in both supervised and unsupervised settings.


To investigate the characteristics of various near work related behaviors among primary students and their associations with changes in myopia related ocular biometric parameters during one-year of follow up.st to 4th grade students aged 6–10 years old were selected by cluster randomization based on probability proportion to size in 2013. At baseline, students together with their parents filled in a self-administered questionnaire on 9 aspects of near work related behaviors and some important covariants of myopia. A comprehensive set of eye examinations including axial length (AL) and cycloplegic refraction was conducted both at baseline and one year later.A school-based sample of 4,814 primary 1P = 0.004], a change in SE  and incident myopia . Selecting an environment with adequate light for visual comfort to read and write was related to elongation of the AL  and increase of AL/CR ratio . Also, not continuing to do near work for more than 30–40 minutes without an eye break was related to increase of the AL/CR ratio  and a change in SE .With the grade level increase, students did increasingly better at finding various ways to have an eye break, but they were increasingly likely to continuously do long-time near work without an eye break. Keeping a reasonable eye distance and correct hand posture for reading, writing, or watching TV became worse for the first time before grade 2, but then became better at grade 3. In contrast, selecting appropriate lighting environments or situations and keeping a balanced diet became better for the first time before grade 2, but then became worse at grade 3. At one-year follow up, the mean AL increased by 0.32 ± 0.35 mm, the ratio of AL divided by the mean corneal radius of curvature (AL/CR ratio) increased by 0.032 ± 0.054, the myopic spherical equivalent (SE) increased by -0.51 ± 0.51 diopters and the incidence of myopia was 16.0% . After controlling for the confounding effects of parental myopia, student’s age, gender, height, daily near work time, daily outdoor activity time and all of the other near work related behaviors, keeping a reasonable distance when reading, writing and watching TV was associated with elongation of the AL [standard coefficient beta = -0.062, Various near work related behaviors changed according to grade level in primary school students. Independent of hereditary factors, daily near work load and outdoor activity, near work related behaviors such as keeping an inappropriate eye distance for near work, selecting inadequate lighting environments, and continuing to do near work without an eye break were risk factors for myopic shifts. Myopic change is becoming a global public health problem with high costs associated with correction and treatment for complications such as myopic macular degeneration, retinal detachment, glaucoma, and cataract . AccordiFactors identified as associated with myopia included parental myopia ,7, outdoThis study was intended to comprehensively and multi-dimensionally explore characteristics of near work related behaviors in primary school students in Shanghai, China and their associations with myopia related oculometric parameters, including both biometry indicators such as axial length (AL) and the axial length-corneal radius (AL/CR ratio), and indicators after cycloplegic refraction, such as spherical equivalent (SE) and incident myopia. To fully understand the causal effects of near work related behaviors on myopia, we analyzed the data of near work related behaviors at baseline (before the progression period) and their associations with myopia progression through a prospective study design.The study was part of a 3-year public health program (2011–2013) to establish archives of the refractive status of children in Shanghai, which was aimed to describe the prevalence of myopia, analyze factors related to myopia and develop personalized assessment tools, develop parameters of refractive development for preschool students and primary school students, draw age specific curves and determine cut-off values for myopia screening and establish three levels of preventive networks for myopia. The project was conducted by the Department of Maternal and Child Health of the School of Public Health of Fudan University, Department of Eye Disease Prevention, Shanghai Eye Disease Prevention and Treatment Center, and the Eye and ENT Hospital of Fudan University from September 2013 to October 2014. The design was a school-based one-year cohort study.The data were from the Elaborative Shanghai Childhood Ocular Refractive Development Study (E-SCORDS) in China. This study was focused on the near work related behaviors and their associations with myopic shifts in primary school students in the Jiading District of Shanghai, China. Based on research by He and colleagues in Guangzhou, the 5-year cumulative incidence of myopia in 6-year-old children was calculated to be 25.7%, and the 2-year cumulative incidence of myopia in 12-year-old children was 31.4% . Sample At baseline (2013), with exclusion of the cases with obvious strabismus (20), cataracts (3), nystagmus (2), and ptosis (2), 4,787 of the 4,814 sampled and registered students were included in the analysis. Among the 4,787 that were planned to complete the questionnaire survey, 4,222 (88.2%) finished the questionnaire regarding basic information, near work time, outdoor activities, and near work related behaviors. Of the 4,222 completed questionnaires, 2,871 (68.0%) agreed to undergo all the examinations including cycloplegic refraction. On the day the examinations were performed, the actual number of students who finished participating in the examinations was 2,840 (67.3%). In 2014, 2,832 (99.7%) of 2,840 students examined at baseline returned for the follow-up examination, whereas only 2,004 (70.8%) students agreed to have cycloplegic refraction, and 1,957 (69.1%) finished cycloplegic refraction .Eye examinations were performed by a clinical team composed of 5 optometrists, 2 public health doctors, 1 ophthalmologist, 1 field assistant, 1 school nurse, and 1 study manager. An experienced public health physician specializing in the prevention of children’s eye diseases from the Shanghai Eye Disease Prevention and Treatment Center was made the project coordinator and ran the entire investigation. All children completed a comprehensive eye examination at both baseline and one-year follow-up.The examination process began with testing visual acuity at 4 m using a retro-illuminated Early Treatment of Diabetic Retinopathy Study (ETDRS) chart with tumbling-Eoptotypes. Acuity was tested with and without refractive correction for those wearing spectacles. Cycloplegic refraction was performed with a Desktop Autorefractor  with a measurement range of -20 to +20 diopters (D). This instrument provided a median value of 3 reliable readings in each eye. The medians were used for the analysis. Each student was reexamined if the differences between the any 2 results of the 3 results obtained were greater than 0.50 D. Cycloplegia was induced in each eye by the instillation of 1% cyclopentolate. Each student was first administered one drop of topical anesthetic agent  to alleviate discomfort, followed by one drop of 1% cyclopentolate (Alcon) after a 15-second interval. One more drop 1% cyclopentolate (Alcon) was given after 5 minutes. Twenty minutes after the last drop, a third drop of cyclopentolate was administered if the pupillary light reflex was still present or the pupil size was less than 6.0 mm . MeasureThe survey was divided into two phases: the school mobilization and questionnaire interview phases. School mobilization was implemented in selected school sampling units by project members and school teachers through a meeting in which the details regarding the questionnaire were explained to the parents and guardians. The mobilization was conducted two to three days before the questionnaire survey. Together with class teachers, the project members gave the students the questionnaire and gave them detailed instructions on how to fill it in with their parents or guardians. Students’ parents or guardians, together with the students, answered the self-administered questionnaire with detailed instructions at home and gave it to the class teachers on the next day. An audit of the questionnaire was conducted to increase its validity, with verification procedures that included checks by class teachers, audits by quality control staff , and phone reviews when necessary.A detailed questionnaire was administered to obtain information on students’ demographic characteristics, students’ activities, and parental myopia at baseline. Students’ activities included near work activities, outdoor activities, and near work related behaviors. Near-work activities were mainly focused on homework-based time (such as exercises or tutoring), and paper-based reading time . The assessment of outdoor time was based on questions about walking, playing outdoors, having picnics, and playing outdoor sports. The above information was obtained by inquiring about the average time spent on such activities during weekdays and weekends separately. The average number of daily near work and outdoor activity hours was calculated using the following formula: [(hours spent on a weekday) × 5 + (hours spent on a weekend day) × 2] / 7. A parent was considered as myopic if he or she used spectacles primarily for distance or for both distance and near.Questions about students’ near work related behaviors in the “last” week were taken directly from the Child Vision Care Behaviors Scale (CVCBS) 18] and  and 18] Before the start of the program, every member of the research team was given the same training regarding the aims, methods, and processes of the investigation. To increase the rate of participation, the details regarding the examination and questionnaire were explained to the parents and guardians of all children at a meeting prior to the examination. Before the survey, we pilot tested and refined the questionnaire’s language, logic, and sequence of questions. During the field data collection, the project members handed out all the questionnaires and gave detailed instructions on how to fill it in. An audit of the questionnaire was conducted to increase its validity, with verification procedures that included checks by class teachers, audits by quality control staff , and phone reviews when necessary.A database was established using Epidata3.1 and double data entry and logic verification were conducted. Then statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) 16.0 .P < 0.001), only data from the right eye were included in analyzing elongation of the AL, increase of the AL/CR ratio and change in the SE. Myopia was defined as SE ≤ -0.50 D. Children were determined to have incident myopia if classified as not myopic at baseline and myopic in either eye at one-year follow-up. The incidence of myopia was defined as the proportion of children who were not myopic (at risk) at baseline and who subsequently developed myopia in either eye during the one-year follow-up period.SE was determined with the following equation: SE = spherical degree + 0.5 × cylinder degree. The AL/CR ratio was defined as the AL divided by the mean corneal radius of curvature. The elongation of AL, increase of AL/CR ratio, change in SE and incident myopia were used to reflect the myopic shifts. As the right eye was closely related to the left eye  and qualitative indicators are described in relative terms. Comparisons of quantitative indicators between different groups were checked with a χork time ,20,21. AThis study was approved by the Ethics Committee of the Shanghai General Hospital, and the research was conducted in accordance with the Declaration of Helsinki. The nature and possible consequences of the study were explained at each school. After the school principal agreed to participate, the details regarding the examination and questionnaire were explained to the parents and guardians of all children at a meeting prior to the examination, and written informed consent was obtained from each. The children provided verbal consent on the day of the examination and survey. Examination after cycloplegia was only performed in children whose parents and guardians had agreed to their participation for all examination items. Children whose parents and guardians had agreed to participation in all examination items, except for cycloplegic refraction, were given an examination without cycloplegia.P = 0.707; CR: t = -0.950, P = 0.342; AL/CR: t = 0.446, P = 0.656; age: t = -0.983, P = 0.326). Girls tended not to complete cycloplegic refraction . The difference between students who completed cycloplegic refraction compared to those who did not undergo cycloplegic refraction at one-year follow up among participants who had completed cycloplegic refraction at baseline was also analyzed. The difference in the CR, AL/CR, and SE values at baseline, and age of students and the gender distribution of the students who completed cycloplegic refraction  were not statistically significantly different from those who had not undergone cycloplegic refraction (883).The difference in the mean AL, corneal radius (CR), and AL/CR ratio values and age of students who completed cycloplegic refraction  were not statistically significantly different from those who did not undergo cycloplegic refraction  . We used the baseline valid questionnaire results and valid information of SE at both baseline and follow-up (n = 1934) to analyze the association between near work related behaviors and changes in SE. In our study, 1,477 were non-myopic at baseline and their data was used to analyze the association between near work related behaviors and incident myopia.Among the 4,129 students who were included in the analysis, 2,220 (53.8%) were boys and 1,909 (46.2%) were girls, aged 8.11 ± 1.17 years (range 6.08–13.17 years). The mean height was 129.51 ± 12.60 centimeters (cm), and the students grew taller as they aged during the year. About half of the parents were both non myopic (47.5%) and 17.5% of parents were myopes. The average time spent on near work was 2.95 ± 1.72 hours per day, which increased as the grade levels increased (grades 1 to 3 changed from 2.39 to 3.38 hours per day) and then decreased (grades 3 to 4 changed from 3.38 to 3.11 hours per day). The average time spent outdoors was 1.29 ± 1.00 hours per day, which decreased as the grade level increased  had elongated by (0.32 ± 0.35 mm), the mean AL/CR ratio (2.96 ± 0.11) had increased by 0.032 ± 0.054, and the mean SE (0.36 ± 1.44 D) had changed by (-0.51) ± 0.51 D. The incidence of myopia in one year was 16.0% .The scores of each special subcategory of near work related behaviors can be classified into four types according to grade level. The score for keeping a reasonable distance from the eye when reading, writing and watching TV, keeping a correct hand posture to hold a pen when writing and keeping correct body posture when reading and writing decreased at first (grades 1 to 2) and then increased (grades 2 to 4), as in the shape of a “U” (“U-Type”). Scores for not reading in situations that can lead to unstable body posture, selecting an adequate lighting environment for visual comfort when reading and writing and keeping a balanced diet increased at first (grades 1 to 2) and then decreased (grades 2 to 4), as in the shape of a “Inverted U” (“Inverted U Type”). The scores for finding various ways to have eye breaks and doing eye exercises according to the right way increased from grades 1 to 4. The scores for not continuing to do near work for more than 30–40 minutes without an eye break decreased from grades 1 to 4. Except for finding various ways to take eye breaks, keeping a reasonable distance when reading, writing and watching TV and keeping correct hand posture to hold a pen when writing, the differences between other special dimensions of near work related behaviors were significant between boys and girls. Boys did better regarding selecting an adequate lighting environment for visual comfort when reading and writing and keeping a balanced diet, while girls did better at not continuing to do near work for more than 30–40 minutes without an eye break, doing eye exercises according to the right way, not reading in situations that can lead to unstable body posture and keeping correct body posture when reading and writing .P = 0.004] and a low score for selecting an adequate lighting environment for visual comfort when reading and writing .To investigate which special near work related behaviors were related to elongation of the AL and increase of AL/CR ratio, we carried out univariate and multivariate analyses for all the students. The results are shown in P = 0.044] and low scores for selecting an adequate lighting environment for visual comfort for reading and writing .  and then decreased/increased (grades 2 to 4). A change in myopia related oculometric parameters, such as AL, AL/CR ratio, SE, and incident myopia, were significantly associated with some special dimensions of near work related behaviors. Keeping an inappropriate distance when reading, writing and watching TV, selecting an inadequate lighting environment and having visual discomfort when reading and writing and continuing to do near work for more than 30–40 minutes without an eye break were risk factors for myopic shifts.In our study, changes in various near work related behaviors by grade level were explored. Each subcategory of near work related behaviors presented various characteristics. Primary student’s near work related behaviors can be classified into four changing types according to grade level. The scores for keeping a reasonable distance when reading, writing and watching TV, keeping a correct hand posture to hold a pen when writing and keeping correct body posture when reading and writing showing as a “U-type” meant the behavior was better at the lower grade level (grade 1) and higher grade level (grade 4). The behaviors being better in grade 1 may be the result of education, since teachers always give instructions on how to develop good habits for reading and writing when primary students enter school. Those three near work related behaviors being better in grade 4 may due to the rich knowledge and improved cognitive ability with growth , which wIn this study, the mean elongation of axial length was 0.32 ± 0.35 mm and the mean increase in the AL/CR ratio was 0.032 ± 0.054. These data were comparable with the results obtained in the study by Guo et al. who found a mean elongation of 0.26 ± 0.49 for AL and 0.03 ± 0.06 for AL/CC in primary students in Beijing, China . In a siIn our study, special near work related behaviors including keeping an inappropriate distance when reading, writing and watching TV, selecting an inadequate lighting environment for visual discomfort when reading and writing and continuing to do near work for more than 30–40 minutes without an eye break were associated with shifts towards myopia. These findings were consistent with some studies ,13,28. TThe characteristics of the above three near work related behaviors associated with myopic shift were varied; i.e., keeping a reasonable distance when reading, writing and watching TV presented as a “U-type,” selecting an adequate lighting environment for visual comfort when reading and writing presented as an “Inverted U Type” and not continuing to do near work for more than 30–40 minutes without an eye break decreased as the grade level increased. As near work related behaviors were modifiable and had effects on myopic shifts, it was better to suggest targeted intervention and focus on specific students through education to promote students developing good behaviors to prevent myopia.The results of our study suggested a new perspective on myopia prevention through comprehensive understanding of the characteristics of near work related behaviors. Since near work related behaviors accompany every student when they study, they do not need extra time and it is easy to get support from school teachers and parents. Moreover, near work related behaviors can be improved through education. Future studies may focus on keeping a reasonable distance when reading, writing, and watching TV, selecting an adequate lighting environment for visual comfort when reading and writing and not continuing to do near work for more than 30–40 minutes without an eye break, and an interventional study should be designed to explore preventing myopia.This study was a school-based one-year cohort study focused on near work related behaviors and their associations with myopic shifts. The outcomes used in analysis included both biometry and cycloplegic refraction, which was rarely reported before, especially in China. We also presented the effect of near work related behaviors on myopic shifts in all the participating students, students completing cycloplegic refraction both at baseline and follow-up and non-myopic students at baseline. This study was the first to comprehensively and multi-dimensionally explore near work related behaviors and associations with myopic shifts. The study was conducted rigorously using unified equipment and a standardized examination process, implementation by experienced public health physicians and collecting data with an entire process of quality control. Eye physicians, data collectors, as well as quality control and other personnel, received training prior to the study. The questionnaire was pilot tested before it was used in fieldwork and its validity and reliability were tested.Nonetheless, the study had some potential limitations. First, the data on the near work related behaviors were self-reported so that estimates of near work related behaviors could be subject to recall bias. An effort was made to reduce recall bias by inquiring about the information of the previous week and asking both students and their parents or guardians. Second, the score of students’ near work related behaviors changed by grade level as reported. Therefore, it is hard to value whether the behavior was unchangeable during the one-year follow-up. However, we conducted the study from September 2013 to October 2014. In fact, all the eye examinations were started from September to October at both baseline (2013) and follow-up (2014) and the questionnaire survey was implemented in September 2013. The period of a grade level starts from September to July of the next year. A student’s academic load, curriculum design, and teachers change little at the same grade level. Therefore, near work related behaviors at baseline can be representative of the whole year. In a future study, it would be better to perform the questionnaire survey at both baseline and follow-up. Third, another possible limitation was the use of subjective measures for near work related behaviors with answers of frequency. However, such methods have also been used in another previous study .In conclusion, various near work related behaviors would change according to grade in primary students. Independent of hereditary factors, the daily near work time and outdoor activity time, keeping an inappropriate distance with the eye when reading, writing and watching TV, selecting an inadequate lighting environment for visual discomfort when reading and writing and continuing to do near work for more than 30–40 minutes without an eye break were associated with a change in oculometric parameters with both biometry and cycloplegic refraction indicating an increase in myopia in Shanghai within a study period of one year. Our longitudinal study provided additional information on the potentially protective role of near work related behaviors on the myopic change towards myopia. Public health care measures are necessary such as that school teachers and parents take account of the potential for urging students to develop good near work related behaviors to prevent the onset and progression of myopia. Future intervention studies may also potentially address near work related behaviors especially for keeping a reasonable distance, selecting an adequate lighting environment and not continuing to do near work for more than 30–40 minutes without an eye break.S1 File(DOCX)Click here for additional data file.


Background. Although binge drinking prevalence and correlates among young people have been extensively studied in the USA and Northern Europe, less is known for Southern Europe countries with relatively healthier drinking cultures. Objective. We aimed at analyzing prevalence and correlates of binge drinking in a representative sample of young adults in Italy. Methods. We conducted a cross-sectional survey among alcohol-consuming young adults. We carried out univariate and multivariate analyses to assess associations between recent binge drinking and candidate variables. Results. We selected 654 subjects, with 590 (mean age: 20.65 ± 1.90) meeting inclusion criteria. Prevalence for recent binge drinking was 38.0%, significantly higher for females than males. Multivariate analysis showed that high alcohol expectancies, large amount of money available during the weekend, interest for parties and discos, female gender, cannabis use, influence by peers, and electronic cigarettes smoking all were significantly associated with recent binge drinking, whereas living with parents appeared a significant protective factor. Conclusions. More than a third of young adults using alcohol are binge drinkers, and, in contrast with findings from Anglo-Saxon countries, females show higher risk as compared with males. These data suggest the increasing importance of primary and secondary prevention programmes for binge drinking. Binge drinking can be described as heavy alcohol use over a short period of time , and it However, studies on binge drinking characteristics and correlates conducted in Southern Europe are sparse –24, thouWe conducted a cross-sectional survey on young adults aged between 18 and 24 years, drawn up according to the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) Statement - Checklist . The EthWe consecutively recruited not abstemious young adults aged between 18 and 24 years. Those who joined the study received an information sheet and signed a written consent. We included only individuals able to sign the informed consent and excluded people who self-reported consumption of alcohol or drugs. The interviewers were students, peers aged between 18 and 24 years, selected from different Schools of Milano Bicocca University, and receiving 10 hrs training for the research project about data collection procedures, including checking eligibility, providing information on the research project, obtaining consent, and distributing and assisting with questionnaires. Questionnaires were administered through a Smartphone application. Subjects who accepted to participate to the study received a €10.00 mobile phone recharge.Recruitment took place in urban locations of Milan nightlife scene, an area of about 1.3 million of inhabitants. We choose areas with a high density of pubs, clubs, discos, or live music events. The recruitment period was between April and July, 2013.Recent binge drinking was assessed by a single question exploring if a consumption of four or five drinks in a row among females and males, respectively, occurred at least once, in the last two weeks. In order to analyze potential factors associated with binge drinking, we identified from scientific literature , 27–30 r(a) Sociodemographic Characteristics. We collected information on age, gender, country of birth, living condition, relationship, educational and employment status, and financial availability for each weekend.(b) Clinical and Individual Characteristics. As there is a strong association between substance-related behaviors and mental disorders [ have you felt depressed or sad much of the time in the past year? We screened for anxiety symptoms in the same manner, asking: have you felt anxious much of the time in the past year? Single-item questions have been used for depressive disorders screening in both general and clinical populations [isorders –33, partisorders , we inveulations , 36, as ulations . In ordeulations , 39, we ulations . The SURulations .(c) Lifestyle Characteristics. First, we explored the interest for joining events and attending recreational settings where it may be easier engaging in binge drinking, such as night parties or discos [r discos , 41. Secr discos , we checr discos .(d) Substance and Alcohol-Related Behaviors. We analyzed substance-related behaviors potentially associated with binge drinking [drinking , 44, coldrinking , 46. Mordrinking , asking drinking  through drinking . The AEQdrinking .t-test was performed for normally distributed continuous data. If normality assumption was rejected for dependent variable distribution, we used nonparametric Wilcoxon-Mann-Whitney test. Chi-square and Fisher's exact tests were used for categorical variables. We identified covariates significantly associated (P < 0.05) with binge drinking for inclusion in subsequent multivariate analyses. Association with binge drinking was shown as odds ratio (OR) with related 95% confidence intervals (CI) and P value. We carried out logistic regressions, controlling for age and sex (as well as adjusting for clusters related to specific place of recruitment) and for variables that were significantly related to binge drinking in the univariate analyses.Statistical analyses were performed using Stata version 10.0 SE. We carried out univariate analyses to identify attributes characteristic of people who binge drink. The normality of continuous data was checked with Shapiro-Wilk's test. Student's We recruited 654 potentially eligible subjects, with 590 (90.2%) meeting our inclusion criteria. No eligible individual refused to participate in the study. The sample comprised 286 males (48.5% of sample) and 304 females (51.5%). The mean age (±standard deviation) was 20.65 ± 1.90, with no significant gender differences. Most of selected subjects were born in Italy, lived with parents, were single in their undergraduate higher education, and not yet in formal employment. Sociodemographical characteristics of the sample are fully described in being in a relationship) to 99.5% .For most of the variables, we had no missing data, although for possibly sensitive items of questionnaires response rates ranged from 96.3% (P = 0.049). Binge drinkers were significantly older than their nonbinge drinking counterpart . People who recently engaged in binge drinking showed higher scores of both impulsivity (P < 0.001) and alcohol expectancies (P < 0.001) according to SURPS and AEQ-AB scales, respectively. Furthermore, depressive disorders were more frequent among binge drinkers than in nonbinge drinking individuals , but no statistical differences were found for what concerns anxiety symptoms . All smoking habits appeared significantly related to a recent binge drinking episode: cigarettes, cannabis, and e-cigarettes were regularly used by 58.0%, 44.2%, and 6.7% of binge drinkers, and by 42.1%, 25.1%, and 2.5% of nonbinge drinkers, respectively. Univariate analyses showed that having a high interest for parties and discos (P = 0.024), having more than €50 available per weekend (P < 0.001), and having most of friends drinking alcohol (P < 0.001) all were significantly associated with a recent binge drinking episode. On the other hand, young people living with parents (P < 0.001), playing sport (P = 0.025), or who claimed to be religious (P = 0.009) were less likely to have been recently engaged in binge drinking. Univariate analyses for categorical and continuous variables are detailed in People with a recent binge drinking were 224 (38.0% of the sample). Rates of a recent binge drinking were significantly higher among females than males , a high financial availability for each weekend (P = 0.041), interest for parties and discos (P < 0.006), female gender (P < 0.001), cannabis use (P = 0.003), influence by peers (P < 0.001), and e-cigarettes smoking (P = 0.047) all were significant correlates of binge drinking. On the other hand, living with parents appeared significantly protective (P = 0.031). Other variables included in the multivariate analysis, such as age (P = 0.127), cigarettes smoking (P = 0.841), impulsivity (P = 0.229), playing sport (P = 0.506), religiosity (P = 0.333), depressive symptoms (P = 0.384), were not significantly associated with the likelihood of a recent episode of binge drinking.We performed multivariate analysis, taking into account age, gender, place of recruitment, and all variables significantly associated with binge drinking at univariate analyses. Results are described in This study describes the prevalence and correlates of binge drinking in a large representative sample of Italian alcohol consuming young adults, recruited in the Milan night scene.Binge drinking prevalence was 38%, significantly higher among females than males, as confirmed by the multivariate analysis. These rates are slightly higher than those reported by previous studies focused exclusively on Italian university students , 22. FurThe main strength of this study is based on representative sampling procedures in natural settings of nightlife scene. Furthermore, any effort to enhance young people participation has been implemented, including peer interviewers, use of smartphone, and financial incentive. This may explain the high response rates. However, also several limitations need to be acknowledged, first the cross-sectional design, which prevents from identifying causal links between binge drinking and related factors. Moreover, in the quick, 10-minute-questionnaire, a number of sensitive correlates  had to be excluded, though the literature has shown relevant correlations with binge drinking , 58, 59.More than a third of young adults using alcohol from our sample were recent binge drinkers. This is slightly higher than rates of other studies from similar geographical areas , 22, rep