Mutation of both REV7-binding sites eliminates the REV3L–REV7 interaction. Invivo complementation shows that both REV7-binding sites in REV3L are necessary for preventing spontaneous chromosome breaks and conferring resistance to UV radiation and cisplatin. This demonstrates a damage-specific function of REV7 in pol ζ, in contrast to the distinct roles of REV3L and REV7 in primary cell viability and embryogenesis.DNA polymerase zeta (pol ζ) is exceptionally important for controlling mutagenesis and genetic instability. REV3L comprises the catalytic subunit, while REV7 (MAD2L2) is considered an accessory subunit. However, it has not been established that the role of REV7 in DNA damage tolerance is necessarily connected with mammalian pol ζ, and there is accumulating evidence that REV7 and REV3L have independent functions. Analysis of pol ζ has been hampered by difficulties in expression of REV3L in mammalian cells, and lack of a functional complementation system. Here, we report that REV7 interacts with full-length REV3L  Although many types of DNA damage cause replication forks to stall temporarily, the DNA polymerases used for semi-conservative replication generally cannot proceed on damaged DNA ,2. A basSaccharomyces cerevisiae, where the catalytic subunit is encoded by the REV3 gene. Yeast Rev3 is associated with a protein encoded by the REV7 gene. In mammalian cells, a homologous catalytic subunit is encoded by the REV3L gene. The REV3L protein of ∼3100 amino acid residues in human and mouse cells is about twice the size of the yeast protein. The closest Rev7 protein homolog in mammalian cells is called REV7 (gene MAD2L2).The biology and biochemistry of pol ζ was examined first in the yeast ; in mammalian cells, the orthologous subunits are designated POLD2 and POLD3  according to the manufacturer's instructions. All cell lines were routinely checked for mycoplasma contamination using the MycoAlert detection kit (Lonza). Cell lines were validated by short tandem repeat (STR) DNA fingerprinting by the Cell Line Identification Core of the MD Anderson Cancer Center. The STR profiles were compared to known ATCC fingerprints (ATCC.org), to the Cell Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808 (http://bioinformatics.istge.it/clima/)  supplemented with 10% fetal bovine serum and penicillin/streptomycin in a 5% CO/clima/)  and to tWe used the following antibodies: ProteinTech , Sigma-Aldrich  conjugated anti-mouse IgG 1:10 000; A0168, HRP conjugated anti-rabbit IgG 1:10 000; A0545, anti-FLAG M2 affinity agarose gel: A2220), Clontech , Cell Signaling Technology  1:1000; #3724, monoclonal anti-His (27E8) 1:1000; #2366), Abcam , GE Healthcare , MBL (agarose conjugated anti-GFP (RQ2); D153-8)REV3L full-length cDNA was obtained in the pUC19M1 vector from Zhigang Wang (The human ang Wang  and subcang Wang  (kindly FH-REV3L 1974–3130 and 2000–3130 fragments were PCR amplified from REV3L/pOZN as a XhoI–NotI fragment with 5′REV3L 1974 (XhoI) primer (5′-CCGCTCGAGGGAGTTGTCAATAAAGGGTCAAG) or 5′REV3L 2000 (XhoI) primer (5′-CCGCTCGAGGCCCCAAGTCGACAACTGGTTC) and 3′pOZ primer to clone into pCDH-EF1α-Flag-HA-MCS-IRES-Puro. GFP-REV3L 1974–1999, 1974–2025 and 1974–2075 expression vector were PCR amplified from REV3L/pETDuet-1 as a XhoI–BamHI fragment with 5′REV3L 1974 (XhoI) primer and 3′ REV3L 1999 (BamHI) primer (5′-CGGGATCCTTAACATTTGCAAGGCATAATCAC), 3′ REV3L 2025 (BamHI) primer (5′-CGGGATCCTTAGGTTTTAGGCAGTTTCTTGGAAC) or 3′ REV3L 2075 (BamHI) primer (5′-CGGGATCCTTAAATCTGAGAATTATCAACATCC) to clone into pEGFP-C1 (Clontech). REV3L 1974–2025 /pEGFP-C1 mutations were introduced using site-directed mutagenesis kits. GST-REV3L 1974–1999 and 1974–2025 fragments were PCR amplified from REV3L/pETDuet-1 as a XhoI–NotI fragment with 5′REV3L 1974 (XhoI) primer and 3′ REV3L 1999 (NotI) primer (5′-TAAAAGCGGCCGCTTAACATTTGCAAGGCATAATCAC) or 3′ REV3L 2025 (NotI) primer (5′-TAAAAGCGGCCGCTTAGGTTTTAGGCAGTTTCTTGGAAC) to clone into pGEX6P-1 . REV3L 1974–2025 /pGEX6P-1 mutations were introduced using site-directed mutagenesis kits. GST-REV3L 1847–1898 fragment was PCR amplified from REV3L/pETDuet-1 as a XhoI–NotI fragment with 5′REV3L 1847 (XhoI) primer (5′-CCGCTCGAGATGTTGACACCAACTCCTGATAGTTC) and 3′ REV3L 1898 (NotI) primer (5′-TAAAAGCGGCCGCTTAGTCATGATCCAACAAAGTTGCC) to clone into pGEX6P-1. His-REV7 expression vector was PCR amplified from REV7 cDNA as a XhoI–NotI fragment with 5′REV7 (XhoI) primer (5′-CCGCTCGAGATGACCACGCTCACACGACAAG) and 3′ REV7 (NotI) primer (5′-TAAAAGCGGCCGCTCAGCTGCCTTTATGAGCGCG) to clone into pETDuet-1  (Novagen). After construction, all expression vectors were confirmed by DNA sequencing. The shREV7 was purchased from Open Biosystems. The shScramble RNA (Plasmid 1864) was purchased from Addgene. Sequences of hairpin used for shRNAs were as follows: REV7 (5′-CCGGCCCTGATTCCAAGTGCTCTTACTCGAGTAAGAGCACTTGGAATCAGGGTTTTT), scramble (5′-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGG-CGACTTAACCTTAGG).Escherichia coli was done as described previously , frozen in liquid nitrogen, thawed on ice and sonicated. After centrifugation 900 μl of 2B  was added to the supernatant and incubated with 10 μl of anti-FLAG (Sigma) or GFP agarose (MBL) for 4 h at 4°C. The bound proteins were washed with 700 μl of 0.1B three times and eluted with 30 μl of 2× sodium dodecylsulphate (SDS) loading buffer . These samples were separated by polyacrylamide gel electrophoresis, transferred to a membrane and detected with the indicated antibodies and ECL reagents .Protein purification from eviously ,26. Immueviously ,28. BrieGST-fusion protein and His-REV7 and Glutathione-Sepharose 4B (GS4B) beads  were incubated at 4°C for 4 h in 500 μl of 0.1B. The beads were washed three times with 0.1B and eluted with 30 μl of 2× SDS loading buffer .REV3L was established using a human-specific Taqman assay (Life Technologies) at the exon 14–15 boundary: Ex14Fwd: 5′-CACCTGGCCTTAGCCCATTAT-3′, Ex15Rev: 5′-CTCTTCTAAGAGTGTCAGTATTACTTCCTTTC-3′ Probe: FAM-MGB-5′-CAACAGAACCAAAAACA-3′. To compare the recombinant expression to that of endogenous mouse Rev3L, we made a set of primers and a probe that would recognize both human and mouse Rev3L and would not amplify any knockout transcript. The primers/probe were at the exon 26/27 boundary: Ex26Fwd: 5′-GTGAATGATACCAAGAAATGGGG-3′; Ex27Rev: 5′-GTGAATGATACCAAGAAATGGGG-3′; Probe: FAM-MGB-5′ 5′-TACTGACAGTA-TGTTTGT-3′. Expression of the endogenous human REV7 was established using MAD2L2 TaqMan Gene Expression Assay . Assay ID was Hs01057448_m1. Gene expression with mouse or human GAPDH as an expression control was measured on an Applied Biosystems 7900HT Fast Real-Time PCR System.Expression of the recombinant human For RNA sequencing analysis, 100 ng of total RNA samples were converted to cDNA using a NuGEN Ovation RNA-Seq System v2 according to the manufacturer's protocol . NuGEN-amplified double-stranded cDNAs were fragmented into ∼180 bp using a Covaris system . Fragmented cDNAs were run on a SPRI-TE library construction system , and during the adaptor ligation step, uniquely indexed NEXTflex adapters  were used for each of the samples to allow for multiplexing. Adapter-ligated libraries were enriched by PCR using a KAPA library amplification kit   and purified with AmpureXP beads (Beckman Coulter). The purified libraries were quantified using a Kapa library quantification kit (KAPA biosystems). The libraries were loaded on cBot  at final concentration of 10 pM to perform cluster generation, followed by 2 × 76 bp sequencing on HiSeq 2000 (Illumina).Rev3L proficient (Rev3L+/−) and deficient (Rev3L−/−) T-antigen immortalized mouse embryonic fibroblasts (MEFs) were as described . Methods of cell infection and selecting stable cell lines were done as described previously . The next day, various concentrations of cisplatin (Sigma) was added to the wells and the cells were incubated for 48 h. Then the cells were lysed in a reagent that luminesces in the presence of ATP , then luminescence was measured using a plate reader (Biotek Synergy II) and normalized to undamaged control. To analyze sensitivity of immortalized MEFs to ultraviolet C 254 nm peak (UVC) radiation, 3 × 10Rev3L-deficient and complemented MEFs, micronuclei formation was measured. The MEFs were plated into an 8-well chamber slide and the following day, were fixed with paraformaldehyde and stained with DAPI. Photographs of the immunofluorescence were taken with a digital camera attached to a Leica DMI6000B microscope. Micronuclei were counted as small, separate DAPI stained foci adjacent to DAPI-stained nuclei. Statistical analysis was done using unpaired t-test with Welch's correction (P < 0.05).To assess genomic instability in the 7 cells were lysed in 500 μl lysis buffer. One hundred microliter lysed sample (2.9 × 106 cells) was mixed with 25 μl 5× SDS loading buffer for a total of 125 μl, yielding 2.3 × 105 cells per 10 μl. The concentration of purified His-REV7 protein from E. coli was measured by the Bradford method. These samples were separated by polyacrylamide gel electrophoresis, transferred to a membrane and detected with an antibody against REV7. The signal intensity was calculated by ImageJ64 software. The signals of 10 and 5 μl whole cell extract indicated 5.4 and 2.7 ng, respectively, resulting in an estimate of ∼ 5.7 × 105 REV7 molecules per 293T cell.Cells were harvested 48 h after transfection (pEGFP vector). 1.46 × 10in vivo. Human REV3L cDNA was inserted into a mammalian expression vector to express N-terminal FLAG-HA tagged REV3L  were expressed in cells Figure . Endogenet al. . Both ϕϕet al. . In the A co-crystal structure shows that specific interactions occur between REV7 and the proline residues of a REV3L 1847–1898 peptide . Residuein vitro after the GST pull-down experiment  binds directly to REV7 independently of the original REV7 binding site within human REV3L . We also quantified REV7 mRNA when REV3L was expressed ectopically . The amount of REV7 mRNA was also unchanged . These data suggest that although REV3L and REV7 protein levels are inversely correlated and that REV3L is stabilized by proteasome inhibition, REV7–REV3L interaction via the two domains studied here is not necessary for proteosomal-mediated degradation of REV3L.We also investigated the impact of REV7 levels on transcription of tor ELK1 . We analRev3L MEF cell lines . The 4A mutant cDNA was unable to rescue the cisplatin or UVC hypersensitivity of −/−Rev3L MEFs . REV7 protein is 4000 times more abundant than REV3L in Xenopus oocytes (5 molecules per cell) . As a result, REV7 is often found in complexes with other proteins . These have a congenital defect in male spermatagonia after birth due to loss of primordial germ cells. The phenotypes of Rev7-null mice more closely resemble mice with defects in cell cycle regulation or particular stress responses (Moreover, disruption of iability . Deletio-antigen . In cont embryos ,49,50 or embryos ,50. MoreBecause REV3L disruption has much more severe consequences for cell proliferation than REV7 disruption, it appears that there is a REV7-independent role for REV3L in embryogenesis and normal cell proliferation. However, we show here that binding of REV3L to two molecules of REV7 is required for maintaining resistance to DNA damaging agents and for limiting spontaneous chromosome breaks in immortalized cells. The present study therefore reveals a separation of function within pol ζ, where both REV3L and REV7 are needed for damage resistance functions, even though REV7 is not necessary for some critical proliferation-based functions.Supplementary Data are available at NAR Online.


Damage to the brain’s white matter is a signature injury of alcohol use disorders (AUDs), yet understanding of risks associated with clinical and demographic characteristics is incomplete. This study investigated alcohol problem severity, recent drinking behavior, and demographic factors in relation to white matter microstructure in heavy drinkers. Magnetic resonance imaging (MRI) scans, including diffusion tensor imaging (DTI), were collected from 324 participants  who reported five or more heavy drinking episodes in the past 30 days. Drinking history and alcohol problem severity were assessed. A common white matter factor was created from fractional anisotropy (FA) values of five white matter tracts: body of corpus callosum, fornix, external capsule, superior longitudinal fasciculus, and cingulate gyrus. Previous research has implicated these tracts in heavy drinking. Structural equation modeling (SEM) analyses tested the hypothesis that, after controlling for duration of alcohol exposure, clinical and behavioral measures of alcohol use severity would be associated with lower white matter factor scores. Potential interactions with smoking status, gender, age, treatment-seeking status, and depression or anxiety symptoms also were tested. Controlling for number of years drinking, greater alcohol problem severity and recent drinking frequency were significantly associated with lower white matter factor scores. The effect of drinking frequency differed significantly for men and women, such that higher drinking frequency was linked to lower white matter factor scores in women but not in men. In conclusion, alcohol problem severity was a significant predictor of lower white matter FA in heavy drinkers, after controlling for duration of alcohol exposure. In addition, more frequent drinking contributed to lower FA in women but not men, suggesting gender-specific vulnerability to alcohol neurotoxicity. White matter damage is a hallmark injury of alcohol use disorders (AUDs), with substantial atrophy found in postmortem and neuroimaging studies , 2. AnimNumerous DTI studies of individuals with AUDs have found white matter abnormality in networks associated with reward and self-regulation –9. WhiteIn a recent study, our group found that neural reactivity to an alcohol taste cue in heavy drinkers was associated with FA of several white matter tracts in frontoparietal and corticolimbic networks . Lower wAlthough white matter abnormality in AUDs is well established, attempts to link it with specific indices of problem severity, drinking behavior, or demographic characteristics have yielded inconsistent results. Effects of heavy alcohol consumption on white matter may interact with gender, age, treatment-seeking status, smoking status, or comorbid psychiatric disorder. Some studies have found greater alcohol-related white matter damage in women , older iThe present study used structural equation modeling (SEM) to test associations of white matter microstructure with clinical and demographic variables. A latent white matter factor (WMF) was created from FA values of the five tracts of interest. This approach had the benefit of limiting the number of statistical tests by reducing several FA values to a single measure of white matter microstructure. In addition, a latent variable isolates variance common to the observed variables and explicitly adjusts for random error, avoiding the problematic assumption of the absence of measurement error in multiple regression , 24. MedThis study investigated three main research hypotheses. First, we hypothesized that a common factor would account for shared variance in FA values of white matter tracts previously implicated in AUD research , within a heavy-drinking sample. This hypothesis was tested using confirmatory factor analysis (CFA) on the WMF model. Second, we hypothesized that, after controlling for duration of alcohol exposure, clinical and behavioral measures of alcohol problem severity would be associated with lower FA. Number of years drinking was chosen as the independent predictor because our theoretical model  stipulatParticipants were ages 21–56 years and reported at least 5 heavy drinking episodes  in the Of the 336 participants in Monnig et al. (2013), 12 had outlier FA values > 3 standard deviations (SDs) from the mean and were excluded from the present study. Consequently, 324 participants were included in confirmatory factor analysis (CFA) of the white matter common factor. SEM path analyses were limited to 303 participants who had data for years drinking. Sample characteristics are shown in Participants completed the Alcohol Use Disorders Identification Test (AUDIT ), the Alr = 0.470, p < 0.001).Age groups were formed using a median split (median age = 27.5 years). Participants were coded as smokers if they reported any smoking on the Timeline Followback. Smokers reported an average of 8.5 ± 7.5 cigarettes per smoking day. A dichotomous variable  was created to indicate the presence of significant comorbid anxiety or depression according to published clinical cutoffs on the BAI and BDI. Participants with BAI score > 15 or BDI score > 13 were coded as high comorbidity. Interest in comorbidity pertained to the presence of common symptomatology rather than either specific diagnosis. For 202 participants with scores for both assessments, the BDI and BAI were moderately correlated  or other formal system was not available for most participants. To facilitate comparison with previous studies of individuals with confirmed AUDs, we report percentages of the sample endorsing high levels of alcohol problems on widely used screening instruments, the ADS and AUDIT. The percentage of the sample meeting the threshold for high probability of an AUD, with a score of 9 or higher, was 64% on the ADS . The per2, 3.5 mm thickness, 1 mm gap). High-resolution T1-weighted MP-RAGE anatomical scans were acquired with TR = 2530 ms, TE = 1.64 ms, flip angle = 7°, 192 sagittal slices, 256x256 matrix, slice thickness = 1 mm). DTI scans were acquired with single-shot, spin-echo, echo-planar imaging along the AC/PC line with FOV = 256x256 mm, 128x128 matrix, slice thickness = 2mm (isotropic 2 mm resolution), NEX = 1, TE = 84 ms, and TR = 9000 ms. A multiple-channel radiofrequency channel coil was used with GRAPPA(X2), 30 gradient directions, b = 800 s/mm2, and b = 0 repeated 5 times. DTI preprocessing steps included quality check to exclude volumes with scanner noise, signal dropout, or excessive motion; motion eddy current correction; and adjustment of diffusion gradient directions. A DTI volume was excluded if the motion was more than 4 mm of root mean square displacement, and a subject’s dataset was excluded if more than 10% of gradient directions were dropped for any reason. All images were registered to a b = 0 s/mm2 image using 12 degrees of freedom, affine transformation with mutual information cost function. Image registration and transformation steps were performed with the FMRIB Software Library (FSL) Linear Image Registration Tool. Outlier detection and data pruning were done with a custom program written in IDL (www.ittvis.com). Dtifit was used to calculate diffusion tensor and FA maps. FA values were obtained using FMRIB’s tract-based spatial statistics , gender , age group , and comorbidity , but not for smoking status. FA was significantly lower in treatment-seeking individuals, women, the older age group, and individuals with comorbid anxiety or depression.Preliminary ANOVAs to test effects of group status on observed FA values were significant for treatment-seeking status [b path; χ2 difference test p-value = .002). The path from PDD to WMF was significant for women  but not for men , meaning that frequency of drinking had a negative effect on WMF scores only in women . Tests of other between-group factors as moderators did not find evidence that mediation effects differed by group.Next, moderation mediation models tested whether mediation effects for ADS or PDD differed by treatment-seeking status, gender, age, smoking status, or comorbidity. Gender significantly moderated the path from PDD to WMF  provided a very good fit to DTI data from these tracts supported this hypothesis. Second, as predicted, individuals with greater alcohol problem severity or drinking frequency had lower white matter FA, after controlling for duration of alcohol exposure. Specifically, problem severity (ADS score) and drinking frequency (recent PDD) significantly mediated the path from number of years drinking to WMF. Third, the effect of PDD on white matter FA differed by gender, such that more frequent drinking was associated with lower FA in women but not in men. Our previous study on this sample of heavy drinkers found that lower FA in these white matter tracts correlated with greater neural reactivity to an alcohol cue. A contribution of the current study is to link drinking behavior, clinical characteristics, and demographic factors to white matter microstructure of these tracts.a priori and data-driven methods. The common factor reflected shared variance in FA values of the tracts used as its indicators and therefore was a specific, rather than global, representation of white matter microstructure. These tracts have been implicated in the pathophysiology of AUDs, and our analysis was a novel approach to investigate associations between white matter microstructure and clinical characteristics in heavy drinkers. Previous neuroimaging studies of AUDs have demonstrated the ability of heavy drinking to damage brain tissue, yet specific markers of risk have yet to be identified [The tracts included in the WMF were selected using a combination of entified . A cliniHigher PDD also was linked with lower white matter FA, and this effect was significantly moderated by gender. Women, but not men, showed a significant relationship between greater drinking frequency and lower white matter factor scores . The modGender also exerted a main effect, with women having lower FA overall. Many DTI studies have reported sexual dimorphism in healthy individuals. Previously, men have shown higher FA in the bilateral tracts included here, whereas women have less consistently shown higher FA in corpus callosum and fornix –49. In aThe main effect of treatment-seeking status on FA values, wherein treatment-seeking individuals had lower FA compared to treatment-naïve individuals, is consistent with previous reports of greater white matter abnormality in treatment seekers . The absSeveral limitations should be considered when interpreting study findings. One reason for using number of years drinking as the independent predictor was to relate historical data to current white matter microstructure. Nevertheless, the cross-sectional study design does not account for the likelihood of premorbid differences or allow for causal inferences. Because number of years drinking was highly correlated with age, we were unable to differentiate the effects of age and chronic alcohol exposure on white matter microstructure. We observed a main effect of age on FA in which the older group (ages 27.5–56) had lower FA values overall. Two large-scale studies of healthy white matter development have placed peak FA at approximately age 30 for most tracts , 42. TheIn summary, this study linked alcohol problem severity to white matter abnormality and provided initial evidence of female-specific vulnerability to frequency of heavy drinking. It utilized advanced statistical modeling to address pressing clinical research questions about alcohol use and white matter microstructure. Future studies might benefit from integrating additional DTI metrics and relating white matter microstructure to gray matter structure and function.S1 File(PDF)Click here for additional data file.S1 Tablep < .05) between groups in a moderator category. Pairwise group differences in FA were not the focus of the current study and therefore were not tested. FA values by moderator group are shown only for descriptive purposes. Measure abbreviations: ADS = Alcohol Dependence Scale; AUDIT = Alcohol Use Disorder Identification Test; ICS = Impaired Control Scale; DPDD = drinks per drinking day; NYD = number of years drinking; PDD = proportion of days drinking; PHDD = proportion heavy drinking days. Tract abbreviations: BCC = body of corpus callosum; CGL = cingulate gyrus, left; CGR = cingulate gyrus, right; ECL = external capsule, left; ECR = external capsule, right; FNX = fornix; SLFL = superior longitudinal fasciculus, left; SLFR = superior longitudinal fasciculus, right.* indicates a significant difference ((PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


The density of functional synapses is an important parameter in determining the efficacy of synaptic transmission. However, how functional presynaptic terminal density is regulated under natural physiological conditions is still poorly understood.2+ concentration was effective in increasing the density of functional terminals. Interestingly, the upregulation was not due to synaptogenesis, but to the conversion of a considerable proportion of presynaptic terminals from nonfunctional to functional. Mechanistic studies revealed that the nonfunctional terminals had inadequate Ca2+-sensitivity-related proteins, resulting in very low Ca2+ sensitivity within their vesicle release machinery. We identified energy-dependent axonal transport as a primary factor controlling the amount of Ca2+-sensitivity-related proteins in terminals. The elevation of intracellular Mg2+ enhanced local energy supply and promoted the increase of Ca2+-sensitivity-related proteins in terminals, leading to increased functional terminal density.We studied the factors controlling the density of presynaptic functional terminals at single dendritic branches of hippocampal neurons and found that elevation of intracellular MgOur study suggests that local energy supply plays a critical role in controlling the density of functional presynaptic terminals, demonstrating the link between energy supply and efficacy of synaptic transmission.The online version of this article (doi:10.1186/s13041-015-0132-z) contains supplementary material, which is available to authorized users. Functional synapses are the elemental unit of synaptic computation in the neural network, and the density of functional synapses determines the capacity for information transmission . InteresGenerally, synaptic silence can be divided into two categories based on the locus of silence, either pre- or postsynaptic. Molecular mechanisms of postsynaptic silence have been studied extensively, determining that lack of AMPA receptors at the postsynaptic locus is largely responsible for postsynaptic silence , 15, 2. Pr) o) from 0.8 to 1.2 mM can efficiently enhance presynaptic plasticity [2+]o could influence functional terminal density. After a long term (48 hr to 2 weeks) increase of [Mg2+]o in culture medium, we found that functional terminal density (N5AP) was positively proportional to [Mg2+]o as [Mg2+]o increased from 0.4 to 1.2 mM. However, to our surprise, N5AP decreased as [Mg2+]o further increased from 1.2 to 2.4 mM , since intracellular Mg2+ is conventionally considered an important endogenous factor involved in multiple intracellular regulations. We used an intracellular Mg2+ indicator, Magnesium Green-AM ester (MgGrn), to label intracellular Mg2+ at basal condition (without eliciting any AP stimulus), the fluorescent intensity of MgGrn could be considered proportional to [Mg2+]i (see Methods). Thus we used MgGrn fluorescence to determine the relative quantity of [Mg2+]i in each branch and to estimate the average [Mg2+]i at a local area of the network (see Methods). By measuring MgGrn fluorescence in the neurons cultured in different [Mg2+]o culture media (for 48 hr to 2 weeks), we found that altering [Mg2+]o led to a dramatic change in MgGrn fluorescence at basal condition  versus [Mg2+]o exhibited a bell-shape relationship  and elevated [Mg2+]o from 0.8 to 1.2 mM for 4 hr (1.2 4hr). We then marked intracellular Mg2+ with MgGrn and functional terminals (responding to 5AP bursts) with FM4-64 and colocalized FM4-64(+) puncta and MgGrn(+) fluorescence at each branch  puncta and MgGrn(+) fluorescence increased  , to rule out the possibility that increasing [Mg2+]o could increase Ca2+ channel blockage, thereby contributing to the increase in functional terminal density. IM, which can increase [Mg2+]i by blocking efflux of Mg2+ through Mg2+ channels [N5AP was increased in concert with the elevation of [Mg2+]i, and the enhancement persisted for ~32 hr  Fig.  and d. I hr Fig.  and e. Schannels , was adm hr Fig.  and f. B2+]i by [Mg2+]o, N5AP could be conveniently and reversibly altered under physiological conditions. Since 4 hr [Mg2+]o elevation was effective at increasing N5AP , we labeled structural terminals with antibodies against several presynaptic proteins, including synaptophysin , VGLUT1 (excitatory vesicle protein), VGAT (inhibitory vesicle protein) and Bassoon (active zone protein)  Fig. . There w hr Fig. , suggest2+]o condition, almost all available structural terminals had the ability to release vesicles under maximal stimulation  was close to that of structure-protein positive puncta (SYP(+) 1.50 and Bassoon(+) 1.47 μm−2), indicating almost all terminals are functional under maximal stimulation  , to deteion Fig. . The denion Fig. .2+]i condition, the 5AP bursting input-induced FM(+) puncta density was remarkably lower . It is possible that this difference was due to the ability of terminals to release vesicles under maximal stimulation but not under 5AP bursting input. To test this possibility, we compared the vesicle release of terminals under 5AP and 600AP stimulations at the same dendrite , raising the [Ca2+]o/[Mg2+]o from 1   induced no increase in N5AP   might lead to the neutralization of the increase in N5AP. Indeed, reducing [Ca2+]o/[Mg2+]o to 0.5 ([Ca2+]o 0.6 and [Mg2+]o 1.2 mM) led to a significant decrease in N5AP in comparison with that under normal [Ca2+]o/[Mg2+]o , there were no differences in N5AP at the low (Ctrl 0.8) and high [Mg2+]o conditions (1.2 4hr).We then determined what cellular processes were modified after elevation of o Fig.  and f, s2+] conditions (e.g. Ctrl 0.8), approximately half of the terminals failed to release vesicles in response to physiological patterns of input and are thereby in a nonfunctional state; (2) the non-function is due to low presynaptic Ca2+ sensitivity, which can be ameliorated by increasing Ca2+ influx by either boosting temporal intensity of stimuli or elevating [Ca2+]o/[Mg2+]o ratio; (3) elevation of [Mg2+]i can enhance presynaptic Ca2+ sensitivity such that most terminals are capable of releasing vesicles to physiological stimuli, leading to higher functional terminal density at dendritic branches.Taking these results together, we concluded that (1) at low [Mg2+]i affects presynaptic Ca2+ sensitivity, we investigated the potential molecular mechanisms underlying this phenomenon. Generally, Ca2+ sensitivity of presynaptic terminals is determined by three biophysical factors  the quantity of Ca2+ sensor proteins  the distance from Ca2+ channels to vesicle release machinery, which determines the coupling efficacy of the Ca2+ influx to the operation of release machinery. The coupling efficacy is regulated by multiple vesicle and active zone proteins  o condition (1.2 4hr) using IF staining. There was no difference in the expression level of Cav2.1 and Cav2.2 per area of dendrites  and coupling proteins  were significantly higher in terminals at the high [Mg2+]o condition  .To address these three possibilities, we compared the profiles of protein expression in terminals at the low (Ctrl 0.8) versus the high i promoted the augmentation of Ca2+-sensitivity-related proteins in terminals. Generally, the quantity of proteins in terminals depends on the three major processes involved in the protein life cycle: (1) synthesis, (2) transport, and (3) degradation, so we investigated if [Mg2+]i affected any of these processes in regard to Ca2+-sensitivity-related proteins in terminals.We carried out further experiments to explore how elevating [Mg2+-sensitivity-related proteins from total proteins extracted from entire neurons at the low (Ctrl 0.8) and high [Mg2+]o (1.2 6 hr) conditions by Western blot. Surprisingly, the quantity of each protein at the high [Mg2+]o condition was not significantly higher (at least within 6 hr) than that at the low [Mg2+]o condition .First, we compared the quantity of SYP and Caion Fig. , ruling 2+]i could promote the transport of Ca2+-sensitivity-related proteins from soma to axonal terminals. Interestingly immunoreactive fluorescence for the Ca2+-sensitivity-related protein Rab3(+) decreased in somatic area  but increased in terminals . This result was also found for other Ca2+-sensitivity-related proteins tested  . The immunoreactivity of SYT1 in the somatic area  Fig.  also had.2) Fig. . These drea Fig. , upper a2+]i on the rate of protein degradation in terminals. Assuming that presynaptic terminals do not have the capacity to synthesize new Ca2+-sensitivity-related proteins (no evidence has been found that they do), the quantity of presynaptic Ca2+-sensitivity-related proteins at equilibrium would be largely determined by the balance of protein transport and degradation. Therefore, if we blocked axonal transport, the rate of protein decline would reflect the rate of protein degradation. When axonal transport was blocked by 0.5 mM Colchicine (Colch) i did not alter the degradation rate of presynaptic proteins, indicating that degradation rate was not part of the mechanism by which increased [Mg2+]i altered presynaptic Ca2+ sensitivity  , the conity Fig.  and f.2+-sensitivity-related proteins had a relatively fast degradation rate and thereby their quantities in terminals at dendrites were strongly dependent upon the efficiency of axonal transport. The elevation of [Mg2+]i likely promoted the augmentation of Ca2+-sensitivity-related proteins in terminals by increasing the efficiency of their transport from soma to terminals, which ensured sufficient Ca2+ sensitivity for AP-dependent vesicle turnover . To validate this, we measured the temporal changes of 5AP Fig. . Further+]i Fig. . These d2+ may determine the efficiency of protein transport in axons. Previous studies indicate that [Mg2+]i and intracellular ATP concentration ([ATP]i) are two endogenous factors critical for controlling the speed of axonal transport [2+]i might improve the efficiency of axonal transport by affecting the efficacy of motor proteins i on the spatial distribution and function of mitochondria. The function of each mitochondrion was quantified by measuring its membrane potential (ΔΨ), using the ΔΨ-sensitive fluorescent dye JC-1 i .We investigated whether the increased quantity of Ca]i Figs.  and d. Mons Fig. . We obseNmito with the quantity of the structure-related protein SYP (QSYP) and with ΣQproteins, we found that ΣQproteins was correlated with Nmito, while QSYP was not , or decreased by either adding FCCP  or by lowering the extracellular glucose concentration (from 28 to 2 mM) for 12 hr in sister cultures from the same batch of cultured neurons, and then [ATP]i, ΣQproteins and N5AP under these conditions were compared. Using cultures under the low [Mg2+]o condition (Ctrl 0.8) as a control, we plotted the relationship between the change of [ATP]i and the change of ΣQproteins and N5AP. The increase or decrease in [ATP]i was linearly correlated with the increase or decrease in ΣQproteins and N5AP  with Magnesium L-Threonate (MgT) in drinking water. This treatment is known to be effective in elevating [Mg2+]CSF CSF . After 8Nmito). We measured Nmito (number of mitochondria per unit area of image) in EM images of each animal  was ~26 % higher than that in control group    Fig. . The extons Fig. .Fig. 8Qu2+-sensitivity-related proteins  in 70 nm ultrathin slices by IF. The total fluorescent intensity of immunoreactive puncta of Ca2+-sensitivity-related proteins was higher in MgT group than that in control group  in each individual rat, and found no correlation between QSYP and Nmito for each rat in both groups  was linearly correlated with Nmito in both groups, and MgT treatment positively shifted the correlation curve  than that in control group, suggesting that the administration of MgT can promote the presence of Ca2+-sensitivity-related proteins in the Stratum Radiatum layer of CA1.Next, we checked the expression of structure-related (e.g. SYP) and Caoup Fig. . We calcups Fig.  lower. Hrve Fig.  upper. Tata Fig. . On aver2+-sensitivity-related proteins relative to the structure-related protein SYP (i.e. ΣQproteins/QSYP), and found that it was linearly correlated with Nmito in both control and MgT groups , here wity Fig.  and [Mg2+]i Fig. , whereasity Fig. . Second,ity Fig.  and f. T2+]o to affect [Mg2+]i, we revealed that very low presynaptic Ca2+ sensitivity was largely responsible for the presence of nonfunctional terminals   on transmitter release  Fig.  and d. Pe review , 64) ande review , 17, 26.nes Fig. , d and fals Fig.  and d. Tnts Fig.  was thatE) control the fraction of synaptic terminals that can participate in synaptic transmission (i.e. N ∝ E)  Fig. .2+-sensitivity-related proteins in terminals. Interestingly, the half-life of Ca2+-sensitivity-related proteins  was shown to be much shorter than that of structure-related proteins (e.g. SYP)   Fig.  and f, ials Fig. , ensurinals Fig.  and d. Htly Fig. , resultiity Fig.  and f anity Fig. . Within ply Fig. . The pos2+]o can concurrently enhance the number and function of mitochondria in distal branches, resulting in the increase of reserved [ATP]i  images for an area of interest (AOI) (59 μm × 59 μm) were taken at 1024 × 1024 pixels with a 0.05754 μm/pixel resolution using a 60X NA1.20 water-immersion objective on a Laser Scanning Confocal Microscopy System FV300 (Olympus) at room temperature , the exceptions were clarified in the following methods. A z-stack of images was taken with 0.5-1 μm steps and compressed at maximal intensity to generate the final image, which was then processed and analyzed in Image-Pro Plus 5.0 (IPP5.0) (Media Cybernetics) and/or an open source software Fiji (Image J). For the measurements of vesicle turnover and protein expression in terminals at dendrites, the AOIs were chosen at the high-density regions of dendritic branches among the “islands” of cell bodies. The general criterion for choosing a “high-density dendritic area” was that most of area in the AOI was covered by neuronal branches. For the fluorescent colocalization experiments (described below), the images from the same AOI were aligned together and registered using the algorithms Rigid Body Registration o in culture medium was adjusted to different concentrations according to experimental designs. In each experiment, sister cultures from the same batch were always used for treatment and control conditions on the same day, and the experimental results were repeated using several different batches.High-density primary cultures of hippocampal CA3-CA1 pyramidal neurons of neonatal ey rats < hr were ΔF) was obtained from the difference of loading (F1) and unloading (F2) images, i.e. ΔF = F1 – F2  at a dendritic branch was defined as the number of 5AP bursting stimulation induced FM(+) puncta (#FM5AP) per unit area of dendritic branch (A), N5AP = #FM5AP/A. Retrograde immunofluorescence (IF) staining of MAP2 (as described below), which specifically presents in dendritic skeleton, was used to label dendritic area and A was calculated from MAP2(+) area. To limit the error of A introduced by the immunoreactivity of MAP2(+), the staining and imaging parameters were rigorously controlled in different batches of IF and the grey-scale histograms of MAP2(+) images were equalized before measuring A. Here, to ensure the equalized MAP2(+) marked area could reflect the real morphology of dendrites, the parameters of histogram equalization were set based on the comparison of MAP2(+) and DIC images. For most experiments, the average N5AP of an AOI was calculated from the total number of FM(+) puncta divided by total MAP2(+) area to reduce the sampling error. Then the mean N5AP of all AOIs (1–5 AOIs/coverslip) was used to represent the average N5AP in a coverslip; for each statistical data point, several coverslips were used and the mean ± SEM of all coverslips was represented . For Fig. N5AP at a single segment of dendritic branch. In general, in an AOI  351–1598 presynaptic functional terminals could be detected by FM dyes for different experimental groups under 5AP bursting stimulation.The functional terminal density (The neurons were fixed in 1X PBS (pH 7.4) containing 1 % Paraformaldehyde (E.M.S.), 0.01 % Glutaraldehyde (Alfa Aesar) and 4 % Sucrose (Amresco) for 1 hr RT. The fixatives were gently washed several times in Tris buffer (pH 7.6), which contained (mM): Tris Base 25, Tris–HCl 25, NaCl 150 (Amresco), and was filtered through 0.22 μm film before use. The coverslips were then permeablized and blocked in freshly prepared blocking solution (using the Tris buffer), which contained 1 % (w/v) BSA (Amresco) and 0.1 % (w/v) Saponin (Sigma) for 1.5 hr RT. Primary antibodies were incubated overnight at 4 °C and probed with CF-dye conjugated secondary antibodies (Biotium) for 1 hr RT. Z-stack images (with 0.5 μm step in z-direction) were taken immediately on FV300, and processed as described above. Specifically, images from 236 μm × 236 μm regions were taken at 2048 × 2048 pixels for Fig. v2.2  1:200; Cav2.1  1:200; Synaptotagmin1 (SYT1)  1:200; Rab3a  1:200; RIM1  1:200; Munc13-1  1:200; ERC1b2 (ELKS)  1:200; Syntaxin1  1:100. CF-dye conjugated secondary antibodies: CF488A 1:400, CF555 1:100–1:200 and CF640R 1:100–1:200 (Biotium).The following primary and secondary antibodies were used : anti-MAP2  1:200–1:400; Synaptophysin (SYP)  1:400; VGLUT1  1:300; VGAT  1:200; Bassoon  1:200; Synapsin1  1:400; Ca2+]o (Ctrl 0.8) coverslip or a high [Mg2+]o (1.2 4hr) coverslip respectively (these two coverslips were sister cultures from the same culturing dish); for each protein, the average protein immunoreactivity values were normalized to the mean of the 9 AOIs of Ctrl 0.8 group. We defined the normalized immunoreactivity per unit area of dendrites as Q index, then ΣQproteins = QSYT1 + QRab3a + QRIM1 + QMunc13 + QELKS + QSyntaxin1.For Fig. 2+ indicator, Invitrogen) for 30 min and washed out for 20 min; both at 37 °C. Fluorescent images of several AOIs in each coverslip were taken on FV300 at RT. In the current study, we measured the MgGrn fluorescence at basal condition (without eliciting any AP stimulus)  at basal condition was usually < 100 nM, the contribution of Ca2+ to MgGrn fluorescence was very small [2+]i. Since the measured MgGrn fluorescence was also dependent on the local volume of branches in network, we built a physical model to describe this phenomenon and derived the calculating formula that corrected the effect of varying local volumes.The mature cultures were incubated in Tyrode solution with 2 μM Magnesium Green-AM  Fig.  to elimiry small . Thus, t2+ ions in cytoplasm. We assumed a cylindrical shape of each neuronal branch  was the spatial distribution function of fluorescence, dv was the differential volume at the point , V’ represented the volume of fluorescence detectable space.Where 2+]i was proportional to integral fluorescence per unit volume of a neuronal branch:Then we derived the relationship as follows based on the assumption that  was the range in z-direction, where fluorescence was detectable, L, d were the length (in x-direction) and width (in y-direction) of the maximal projection of fluorescence on xy-plane  as the resolution in x- and y-direction, which was 0.05754 μm/pixel in our experiments, and set ρ as the resolution in z-direction, which was equal to the scanning step of z-stack, i.e. ρ = 1 μm. Thus L = lθ, d = mθ, 2h = nρ, where l, m were the pixels of the branch in x, y directions, n was the number of images in the stack. Then we got:We set By approximate numerical integration, we derived:z) in z-stack .Where 2+]i was approximately positively proportional to From the formula, we knew that i in the branch. The mean value of these 50–100 branches was calculated to represent the level of [Mg2+]i in the AOI. For each coverslip, several AOIs were measured and the mean value of these AOIs was calculated to represent the average level of [Mg2+]i in the coverslip. For each data point, several coverslips were used. For each statistical data point, the mean ± SEM of all coverslips was presented. Specifically for Fig. 2+]i (MgGrn fluo. a.u.) value in an individual branch.In the experiments, given a randomly selected AOI of dendritic area (the image was obtained as described above), 50–100 branches were randomly selected from each AOI and the mean intensity of fluorescence (i.e. ΔΨ) of each mitochondrion was estimated by the ratio of fluorescence of aggregate versus monomer form of JC-1, i.e. Faggr/Fmono [Faggr/Fmono) for each individual mitochondrial punctum to represent the ΔΨ of that mitochondrion. To estimate the valid range of Faggr/Fmono, we measured Faggr/Fmono of individual mitochondria before and after administering 5 μM FCCP 5 min at the same AOI. The mean Faggr/Fmono of all the mitochondria  was lowered to 0.26 ± 0.002 . Thus, we considered the Faggr/Fmono value of a mitochondrion to be meaningful only if it was > 0.26 in our experiments, and the puncta with Faggr/Fmono < 0.26  were excluded in statistics. Faggr/Fmono was calculated for each individual meaningful punctum and the mean value of population was calculated from all the puncta to represent the general ΔΨ in this AOI. 3–5 AOIs were measured for each coverslip and the mean of the AOIs were calculated to represent the average ΔΨ of the coverslip. For each bar or point in Fig. Nmito) by counting the number of JC-1-aggregate(+) fluorescent puncta  for 6 hr. Proteins were extracted from the cultures and then resolved on Polyacrylamide gels. Proteins were transferred from gels to PVDF membrane and probed with the following antibodies: anti-SYP (Millipore) 1:50,000, SYT1 1:5,000, Rab3a 1:500–1,000, RIM1 1:800, Munc13-1 1:400, ERC1b2 (ELKS) 1:1,000, Syntaxin1 1:500–1,000 (SYSY), GAPDH 1:1,000 (CST). The other procedures were the same as previously described [[Mgescribed . For the4. The solution was repeatedly pipetted to ensure complete lysis. The lysed neurons were immediately transferred into a centrifugal tube and centrifuged at 12,000 rpm at 4 °C for 5 min. For each coverslip, total ATP content of supernatant was immediately determined using a Luciferin-luciferase ATP Assay Kit (Invitrogen). For each data point in Figs. Cultured neurons were quickly detached from a coverslip with 500 μL boiling extraction solution  containiSprague–Dawley rats  were fed Magnesium L-Threonate  (Magceutics Inc.) in their water for 8 months [2 square blocks which were then used for electron microscopy (EM) and IF separately. The blocks were rinsed in 1X PBS (pH 7.4) for 1 hr and 1X Maleate Acid (Sigma) buffer (pH 6.0) for 1 hr to wash out the fixatives.Mature 8 months . ControlFor the blocks prepared for EM, they were post-fixed in 1 % Osmium Tetraoxide (Ted Pella) and 1.5 % Potassium Ferricyanide (Sigma) in MB (pH 6.0) for 1 hr, and stained with 3 % Uranyl Acetate (Ted Pella) for 1 hr. Then the blocks were dehydrated with 50, 70, 80, 95 %, 100 %, and 100 % Ethanol sequentially, for 15 min each. Following dehydration, blocks were placed in 100 % Propyleneoxide (Sigma) for 10 min twice. All the procedures above were performed on ice. Then blocks were infiltrated with 50 % Propyleneoxide + 50 % SPI-PON 812 resin (SPI-CHEM) for 60 min RT, 100 % resin for 24 hr at 4 °C, and subsequently transferred into embedment molds fulfilled with pure resin and placed in 60 °C oven for polymerization for 24 hr. 70 nm ultrathin slices were cut and then stained with 3 % Uranyl Acetate and 0.4 % (w/v) Lead Citrate, sequentially. Images were taken on Transmission (Hitachi H-7650)  or Scanning EM  . For the SEM, ultrathin slices were mounted on silicon chips, which can enhance the electro-conductivity dramatically (even without carbon coating). The density of mitochondria in an EM image was defined as #mitochondria/area. For each rat, the density of mitochondria was calculated from the mean density of 67–96 TEM images and 5 SEM images (the density measured in TEM images was the same as that measured in SEM images).F) of all the puncta per area of image was measured to represent the quantity of that protein. For each rat, F values from 5–7 tissue blocks were averaged to represent the protein quantity in that rat (Frat). We normalized Frat of each protein by the mean value of all rats in control group to get the normalized quantity Q. To estimate the total quantity of the Ca2+-sensitivity-related proteins, we summated the Q of the 6 proteins in the same rat together to represent the total quantity in that rat (ΣQproteins), ΣQproteins = QSYT1 + QRab3a + QRIM1 + QMunc13 + QELKS + QSyntaxin1.For the blocks prepared for IF, they were treated with 1 % Tannic Acid (Sigma) for 1hr, 1 % Uranyl Acetate in Maleate Acid buffer (pH 6.0) for 1hr, and then dehydrated with 50, 70, 80, 95, 100 %, and 100 % Ethanol containing 1 % PPD (Sigma) sequentially, for 15 min each. Mixture of Ethanol and LR White resin (E.M.S.) with the ratios 2:1, 1:1, 2:1 and pure resin were used to infiltrate the tissue blocks, each for 1 hr, then replaced by 100 % LR White for 24 hr. All the above procedures were performed on ice. The blocks were placed in capsules filled with LR White resin and put in 55 °C oven for polymerization for 48 hr. 70 nm ultrathin slices were cut and mounted on coverslips. The IF was performed according to Array Tomography technique . SYP, VGAll the rats involved in this paper were purchased from Vital River Laboratory (Beijing). All the animal experiments were approved by Tsinghua University Committees on Animal Care.p < 0.05. Two-tailed Student-t or Kolmogorov-Smirnov test is used.Data are shown as mean ± SEM. Statistical significance is considered as


Amphimedon queenslandica.The aquiferous body plan of poriferans revolves around internal chambers comprised of choanocytes, a cell type structurally similar to choanoflagellates. These choanocyte chambers perform a range of physiological and developmental functions, including the capture of food and the generation of stem cells. Despite the increasing interest for choanocytes as sponge stem cells, there is limited knowledge on the development of choanocyte chambers. Using a combination of cell lineage tracing, antibody staining and EdU labeling, here we examine the development of choanocytes and the chambers they comprise during metamorphosis in the marine demosponge Lineage-tracing experiments show that larval epithelial cells transform into mesenchymal pluripotent stem cells, resembling archeocytes, within 24 h of initiating metamorphosis. By 36 h, some of these labeled archeocyte-like cells have differentiated into choanocytes that will form the first postlarval choanocyte chambers. Non-labeled cells also contribute to these primary choanocyte chambers, consistent with these chambers being a chimera of multiple transdifferentiated larval cell types and not the proliferation of a single choanocyte precursor. Moreover, cell proliferation assays demonstrate that, following the initial formation of choanocyte chambers, chambers grow at least partially by the proliferation of choanocytes within the chamber, although recruitment of individual cells into established chambers also appears to occur. EdU labeling of postlarvae and juveniles reveals that choanocyte chambers are the primary location of cell proliferation during metamorphosis.A. queenslandica chambers can also divide, with choanocyte chambers being the primary location of cell proliferation. Interestingly, the level of cell proliferation varies greatly between chambers and appears to be contingent on the size, location and developmental state of the chamber. Small chambers on the periphery of the body tend to possess more dividing cells. As choanocytes can also dedifferentiate into archeocyte-like cells, cell proliferation in chambers may not only contribute to chamber growth and self-renewal but also increase the number of pluripotent archeocytes.Our results show that multiple larval cell lineages typically contribute to formation of individual choanocyte chambers at metamorphosis, contrary to previous reports in other species that show sponge choanocyte chambers form clonally. Choanocytes in postlarval and juvenile  Sponges are sessile, aquatic animals that possess internal ciliated choanocyte chambers that pump seawater through an aquiferous network of canals, which connect with the external environment through a huge array of pores and enable the capture of food and the removal of waste , 2. As sSponges have remarkable regenerative abilities and the capacity to continually remodel their body shape. These abilities appear to be largely achieved through their two major stem cell types, archeocytes and choanocytes , 19, 20.Given the importance of the choanocyte in the functioning, maintenance, regeneration and repair of the sponge body plan as well as their similarity with choanoflagellates, we sought to determine the ontogenetic origin of this cell type and the chambers they construct. In many demosponges, choanocyte chamber formation is thought to occur by differentiation of a single archeocyte into a choanocyte progenitor cell, which then proliferates to form a mature choanocyte chamber –33. In dAmphimedon queenslandica. Although it is known in this species that larval epithelial cells transdifferentiate into choanocytes and other cell types at metamorphosis [Amphimedon at about 36 h after the initiation of metamorphosis. The number and size of these chambers continue to grow, and at around 72 h after the initiation of metamorphosis, a functional aquiferous system forms. Cell-tracing experiments reveal that choanocyte chambers often form by contributions from multiple larval cell lineages and proliferation of choanocyte progenitors. Continuous proliferation and late recruitment of individual choanocytes contribute to the further growth of these chambers. These results demonstrate that in Amphimedon and potentially other sponges, choanocyte chambers are not always clonal.Here, we use cell lineage tracers, antibodies and cell proliferation markers to follow the ontogeny of choanocytes and choanocyte chambers during metamorphosis in orphosis , 36, 42,A. queenslandica were collected and maintained in flow-through aquaria at the University of Queensland Heron Island Research Station (Great Barrier Reef Marine Park Authority permit G12/35053.1). Larval collection followed the protocol of [Adult tocol of  where adtocol of .Amphiroa fragilissima, which strongly induces settlement and metamorphosis [A. fragilissima were removed using fine forceps  and resettled on to round coverslips placed in a well with 2 ml FSW in a 24-well plastic plate, with 3 postlarvae placed on each coverslip. These resettled postlarvae ball up and take the form similar to a newly settled larva. In terms of recording the time points of metamorphosis, we used this placement of newly settled postlarvae on the coverslips as the starting point of metamorphosis referred to as the 0 h postresettlement (hpr) stage, although they had originally settled on A. fraglissima up to 4 h before this time. Metamorphosis from a resettled larva to a functional juvenile takes approximately 72 hpr [Competent larvae were placed in 6-well plates with 10 ml of 0.2-μm filtered seawater (FSW) for 4 h in the dark with live coralline algae orphosis . After 4y 72 hpr , 42.Postlarvae and juveniles on the coverslips were fixed according to . ImmunohThe lipophilic cell tracker CM-DiI (Molecular Probes C7000) was used to label ciliated epithelial cells as described in . CompeteTo visualize cell proliferation, the thymidine analogue EdU  was used as previously described . Early pOne of the distinct morphological features of choanocytes is the apical flagellum or cilium Fig. . To visuA. queenslandca. Larval epithelial cells were labeled with CM-DiI and were traced through metamorphosis to investigate the clonality of postlarval choanocyte chambers. Only the epithelial layer has CM-DiI-labeled cells , CM-DiI-labeled choanocyte chambers can be observed  antibody that labels mitotic cells  and antiAmphimedon may be dependent on size, location and developmental state.Metamorphosis is complete at about 72 hpr when a functional aquiferous system is established . At thisA. queenslandica. This process begins with ciliated larval epithelial cells and probably other larval cell types transforming into pluripotent archeocyte-like cells within a few hours of the larva first settling and commencing metamorphosis. Archeocyte-like cells appear to be the prevalent cell type during the first 24 h of metamorphosis, suggesting most larval cells that are maintained during this transition transform into archeocytes [Amphimedon can be comprised of cells from multiple larval origins. There were also chambers that were completely labeled with CM-DiI, indicating that these chambers were either generated clonally from a single progenitor cell or formed from multiple cells that were labeled with CM-DiI. The sources of non-labeled choanocytes include archeocytes from the larval inner cell mass [In this study, we investigated the formation and growth of choanocyte chambers during metamorphosis of the demosponge heocytes . Some ofell mass .Ephydatia fluviatilis. In this case, choanocyte chambers form clonally from the proliferation of a single archeocyte precursor [E. fluviatilis. During this process in A. queenslandica, it appears that at least some primary chambers are not derived from a single progenitor cell; some chambers may be clonally derived. Recognizable choanocyte chambers first appear around 36 hpr. This corresponds to a period of a marked increase in cell proliferation, which is enriched in areas where chambers appear to be forming. Chamber growth continues through the formation of the functional juvenile aquiferous system, which occurs at about 72 hpr.Despite the importance of choanocytes in many aspects of a functional sponge, including the stem cell system, the ontogeny of choanocyte chambers during metamorphosis has not been studied in great detail in demosponges. One of the best-described cases of choanocyte chamber development in demosponges is during asexual reproduction in recursor , 48. HowA. queenslandica chambers is a contributing factor to the growth of the chamber, as observed in other sponges [A. queenslandica there is also evidence that chamber growth occurs by recruitment of individual choanocytes. Individual CM-DiI-labeled choanocytes that are not associated with a chamber are observed in the Amphimedon postlarvae and juveniles  initial chambers that are either clonal or cell lineage chimeras, (2) localized proliferation in new and established chambers and (3) late recruitment of individual choanocytes into established chambers. In the first and third cases, choanocytes are derived from progenitor cells, which themselves originate by the transdifferentiation of larval cells early in metamorphosis or from archeocytes in the larval inner cell mass . Althougsms Fig. .Fig. 9PrA. queenslandica, comprised of cells that can undergo self-renewal and dedifferentiate into pluripotent archeocytes [E. fluviatilis [A. queenslandica, choanocytes in juvenile chambers dedifferentiate into archeocytes that can give rise to a diversity of somatic cell types [The high level of proliferation in choanocyte chambers in juveniles is consistent with this being a stem cell niche in heocytes . These aheocytes , 27, sugviatilis , 49. In ll types . CurrentA. queenslandica postlarvae and young juveniles, choanocytes in chambers can be highly proliferative and are capable of dedifferentiating into archeocytes , consiss study; , 50. Alts study; , the spes study; , 26. TheA. queenslandica. This is in contrast to other demosponges, where individual choanocyte chambers appear to be clonally derived from a single progenitor cell. Proliferation of choanocytes in the chamber and recruitment of individual choanocytes from outside the chamber appear to be the primary means by which newly established chambers grow in A. queenslandica, with choanocytes commencing mitosis about 30 h after the initiation of metamorphosis. The choanocyte is perhaps the most proliferative cell type in postlarvae and juveniles, although the level of cell proliferation varies greatly between chambers and appears to be contingent on the size, location and developmental state of the chamber. As choanocytes can also dedifferentiate into archeocyte-like cells, cell proliferation in chambers may not only contribute to chamber growth and self-renewal but also the replenishment of pluripotent archeocytes.This study reveals that multiple larval cell lineages contribute to the initial formation of individual choanocyte chambers at metamorphosis in


This study uses data from a nationally representative household survey  to describe patterns of alcohol consumption and related behaviors among Brazilian adults. Analyses include descriptive and multivariable Poisson regression for self-reports in the past 30 days of: drinking any alcohol, binge drinking, binge drinking 4 or more times, and driving after drinking (DD); as well as age of alcohol consumption initiation. Results show that current drinking prevalence was 26%, with an average age of initiation of 18.7 years. Binge drinking was reported by 51% of drinkers, 43% of whom reported binge drinking 4 or more times. Drinking and driving was reported by nearly one quarter of those who drive a car/motorcycle. Current drinking was more likely among males, ages 25–34, single, urban, and those with more education. Binge drinking was more likely among males, older age groups, and people who started drinking before 18. Drinking and driving was higher among males, those with more education, and rural residents. Those who binge-drink were nearly 70% more likely to report DD. All behaviors varied significantly among Brazilian states. Given their potential health consequences, the levels of injurious alcohol behaviors observed here warrant increased attention from Brazilian policymakers and civil society. Alcohol consumption is associated with increased risks for a number of chronic and acute conditions as well as traffic- and violence-related injuries and deaths –3 While Brazil has the world’s fifth largest population and has experienced considerable economic growth over the past decade. As a rapidly ageing middle-income country, it faces the double burden of high rates of infectious diseases alongside climbing rates of non-communicable conditions and injuries. Of the lei seca in Portuguese) establishing a 0.02% blood alcohol content (BAC) legal limit—equivalent to about 1 beer—for all drivers and imposing considerable penalties for BAC levels of 0.06 or above[Given the high rates of traffic-related fatalities in Brazil, alcohol involvement in motor vehicle crashes bears investigating, especially in light of the country’s “dry law” . Behaviors such as heavy episodic (binge) drinking  are strong predictors of alcohol-related mortality, morbidity, and problematic behaviors . Within Demographic characteristics, such as sex and age, are also associated with drinking behaviors, and these relationships also vary among countries. In most studies, men are more likely to be drinkers and to exhibit problem drinking than women, although as countries move closer to gender equality, women begin to exhibit drinking patterns closer to those of men . YoungerGiven the complexity of the changing social, economic, and demographic situation in Brazil, this study explores drinking patterns using the most recent nationally representative household survey. The purpose is to describe current drinking patterns within the Brazilian population aged 18 and over, identify the prevalence and correlates of problem drinking behaviors, and to assess the relationship between these behaviors and self-reported driving after consuming alcohol.Pesquisa Nacional de Saúde) a nationally representative household survey conducted by the Brazilian Institute of Geography and Statistics (IBGE) and Ministry of Health in 2013. The survey employs a complex sampling design. The primary sampling units are census tracts based on the 2010 census and randomly selected from the IBGE national master sampling plan. Within each census tract households were randomly selected from a national registry of addresses. Within selected households a randomly selected respondent aged 18 or over was invited to take part in the study. The final sample size was 62,986 with an overall response rate of 78% [Data are derived from the National Health Survey . People aged 25 to 34 were more likely to binge-drink than younger respondents (p<0.01), while older respondents (55–64 and 64 and older) were less likely to binge-drink (p<0.001). However, among binge drinkers, the group aged 55 to 64 had the highest prevalence ratio  of binge-drinking 4 or more times in the previous month (p<0.01). College educated drinkers were significantly less likely to binge-drink (p<0.001) and to binge-drink 4 or more times in the past month (p<0.05), compared to those with less than a high school education. Blacks and Asians were respectively 23% and 22% more likely to binge-drink as compared to whites (p<0.001), but only Asian binge drinkers were more likely to binge-drink 4 or more times in the past month compared to whites (p<0.01). Being married or cohabitating was negatively associated with both binge drinking outcomes, as was rural residence, while living in a state capital was positively associated with both. Alcohol initiation before 18 years of age was also significantly associated with binge drinking outcomes: early age drinkers were 23% more likely on average to binge-drink (p<0.001) and these binge drinkers were 16% more likely to binge 4 or more times in the past 30 days than those who reported initiating drinking after age 18 (p<0.01).Factors positively associated with reporting drinking and driving were somewhat different from those predicting binge drinking behaviors (last column). Among drivers, people in both the age groups 25–34 and 35–44 were over 50% more likely to drive after drinking than those aged 18 to 24 (p<0.001). The college educated were 41% more likely to report driving after drinking compared to those with a high school education (p<0.001), and rural residence was positively associated with drinking and driving (p<0.01). Factors negatively associated with self-reported drinking and driving include being female, being married or partnered, and living in a capital city. Among drivers, binge drinkers were 70% more likely to drive after drinking than non-binge drinkers (p<0.001).Despite a prevalence of alcohol abstinence in Brazil that is significantly higher than that of the United States and most wealthy OECD countries, this study has identified high rates of injurious drinking behaviors among those who do consume alcohol . More thOur findings provide a national context for city level differences between men and women observed in several studies –35. WomeThose who described themselves as married or partnered were less likely to report problem drinking behaviors, a finding consistent with other international studies ,38,39. IIn contrast to age-related risk behaviors observed in other countries, the likelihood of driving after drinking was higher among adults ages 25–54, than among those ages 18–24. This difference may be the result not only of the lower likelihood of those 18–24 to be drinkers, but also reflect lesser availability of cars for younger drivers. This situation may very well change with Brazil’s rapid changing economic and social context . Indeed,In this study, initiating drinking before age 18 was associated with a higher likelihood of binge drinking overall and binge drinking more than 4 times a month, and binge drinkers were more likely to report driving after drinking. Our findings are consistent with recent findings that among students aged 14–18 years in the 27 Brazilian state capitals, 30% of youth reported binge drinking behavior in the past year, and those with higher socioeconomic status had higher risks of binge drinking . Early dOther studies have found racial and ethnic disparities in regards to problem drinking and this study underscored such findings. Compared to those who identified as white, Asian and mixed (pardo) individuals were less likely to be drinkers. Among drinkers, blacks and Asians were considerably more likely than whites to report binge drinking, and Asians more likely to report frequent binge drinking, even after controlling for demographic and socioeconomic characteristics. These differences merit further study since interventions to reduce problem drinking in Brazil may require adaption to different social and cultural contexts .Our study provides the first picture of patterns of multiple drinking behaviors across Brazilian states and the geographic differences point to areas where alcohol control efforts should be targeted. Rural residence was associated with an increased likelihood to drive after drinking and we note high rates of self-reported drinking and driving in states with large semi-urban and rural areas. The substantive variation across states in the portion of the population that reports binge-drinking, frequent binge-drinking and drinking and driving bears further investigation. This variation may reflect differences in state populations by age distribution, levels of education, and economic factors such as car ownership. They may also suggest differences in enforcement of existing policies controlling alcohol and impaired driving.Other studies have found self-reports to be underestimates of impaired driving . Our stuThe patterns of alcohol consumption reported in this study warrant attention from Brazilian policymakers and civil society. Among drinkers, the prevalence and frequency of binge drinking and drinking after driving far exceed rates in other industrialized nations. For example, in the United States, a country with relatively high rates of binge drinking only 30% of drinkers report binge drinking. Motor vehicle crashes continue to be a leading cause of death and disability in Brazil with estimates of over 40% of traffic deaths attributed to alcohol involvement . AlcoholThis study has some limitations. Results are based on self-report and are likely to represent underestimates of true values, especially with regard to socially sanctioned or illegal activities such as drinking and driving. However, the large sample size and state-level estimates have been able to illuminate the socio-economic, demographic, and geographic determinants of alcohol consumption and associated risk factors in a large, diverse middle income country. Because the data are cross-sectional we are unable to determine causal relationships and directionality of the observed associations. The results nevertheless point to a cluster of factors associated with elevated risky alcohol behaviors that may be targets for more detailed investigation and intervention.S1 Table1Defined as reporting having at least 1 drink in the past 30 days. 2At least 5 (for men) or 4 (for women) drinks in one occasion among current drinkers. 3 Among those reporting binge drinking in the past 30 days. 4 Among current drinkers who drive a car or motorcycle.Data source: PNS 2013, authors’ calculations. Results include sample weights and control for survey design. (DOCX)Click here for additional data file.


Presbyopia is age-related loss of accommodation that gradually impairs near vision. Few studies have examined the burden of presbyopia in the United States of America (USA)In our study, individuals ≥35 years of age who attended an employment fair sponsored by the Illinois Department of Employment Services (IDES) in the Pilsen neighborhood of Chicago were invited to undergo testing of their near vision and to befitted with reading spectacles, if needed. Individuals with bilateral visual impairment or blindness, as determined by both a history and a screening examination with a pen light  were given information and instructed to seek an eye examination. In the remaining individuals, near acuity was measured at 40 cm, and 133 people were identified as having functional presbyopia .‘Employment status correlated with the frequency with which patients accessed an eye care professional’The 133 respondents ranged in age from 36–85 years old. Their average age was 55 years and 57% were female (74 out of 133). Over 70%  were unemployed. The average uncorrected near acuity was 20/50 (6/15). After receiving reading spectacles, 80% achieved a near acuity of ≥20/25 (6/7.5). Uncorrected near acuity <20/50 (6/15) was associated with greater difficulty reading print (p<0.02) and perceived worse eyesight (p<0.0001) compared to those with uncorrected near visual acuity of ≥20/50.More than 98 per cent of respondents stated they could read. An improved ability to read was cited as the most important benefit of reading spectacles, with minimal impact perceived on other daily tasks Table . Those wWith regards to barriers to obtaining near vision correction, 49 percent (n=64) of the respondents said that the cost of spectacles was a disincentive and 48 per cent (n=63) said that the lack of availability of an eye care professional greatly impeded them from obtaining spectacles Table . ConsideOver two-thirds of the respondents (68 percent) had been examined by an eye care professional at some point in their lives, but only 30 per cent (n=40) had been examined in the previous year. Our population was predominantly Hispanic and African American. Considering that the rates of blindness and visual impairment in these groups is significantly greater than that of non-Hispanic whites,Employment status correlated with the frequency with which patients accessed an eye care professional. Whereas almost 80 per cent of respondents who were employed had seen an eye doctor, less than 64 per cent of respondents who were unemployed had similarly accessed care (p<0.05).In a country where services are readily available and spectacles are affordable, nearly half of the participants were not aware that this was case. All of the participants would have benefited from a pair of reading spectacles.Our study is the first to examine how presbyopia affects a resource-poor population in the USA. The survey highlights the need for continued efforts to ensure that economically disadvantaged and minority populations gain access to eye care services and utilise them. Practical solutions include outreach activities and education of health care providers in low-resource communities about the ease with which their patients may remedy their prebyopic needs with inexpensive, over-the-counter, ready-made reading spectacles. Health care providers should also be educated about the increased rates of blindness and visual impairment in the African American and Hispanic populations so that they can encourage and refer individuals in these population groups for regularly scheduled eye examinations.


Though often used to control outbreaks, the efficacy of ward closure is unclear. This systematic review sought to identify studies defining and describing ward closure in outbreak control and to determine impact of ward closure as an intervention on outbreak containment.We searched these databases with no language restrictions: MEDLINE, 1946 to 7 July 2014; EMBASE, 1974 to 7 July 2014; CINAHL, 1937 to 8 July 2014; and Cochrane Database of Systematic Reviews, 2005 to May 2014. We also searched the following: IndMED; LILACS; reference lists from retrieved articles; conference proceedings; and websites of the CDCP, the ICID, and the WHO. We included studies of patients hospitalized in acute care facilities; used ward closure as a control measure; used other control measures; and discussed control of the outbreak(s) under investigation. A component approach was used to assess study quality.We included 97 English and non-English observational studies. None included a controlled comparison between ward closure and other interventions. We found that ward closure was often used as part of a bundle of interventions but could not determine its direct impact separate from all the other interventions whether used in parallel or in sequence with other interventions. We also found no universal definition of ward closure which was widely accepted.With no published controlled studies identified, ward closure for control of outbreaks remains an intervention that is not evidence based and healthcare personnel will need to continue to balance the competing risks associated with its use, taking into consideration the nature of the outbreak, the type of pathogen and its virulence, mode of transmission, and the setting in which it occurs. Our review has identified a major research gap in this area.The online version of this article (doi:10.1186/s13643-015-0131-2) contains supplementary material, which is available to authorized users. Clostridium difficile continues. In the USA, the prevalence rate of HAI was 4 % in 2011 [C. difficile and vancomycin-resistant Enterococci infections increased from 2007 to 2012, and carbapenemase-producing organisms appeared in 2010 [C. difficile-associated diarrhea is estimated to be at least $128,200 CDN per year per facility [While significant progress has been made in preventing device and procedure-related healthcare-associated infections (HAI), the threat of antimicrobial resistant organisms (ARO) and  in 2011 . An esti in 2010 . The cosfacility . These oOutbreaks of HAI in healthcare facilities are not only serious clinical events when affecting vulnerable patient populations but are highly disruptive to care delivery. Closure of affected clinical areas typically involves suspending new patient admissions and has been used as a means of controlling HAI outbreaks . HoweverComplete ward closures are typically exercised when other outbreak measures have failed, or in the setting of highly virulent organisms, or those known to spread rapidly . HoweverA number of studies have described the use of ward closure for the purpose of outbreak control. One systematic review of worldwide HAI epidemics published in the Worldwide Database of Nosocomial Outbreaks between 1965 and 2005 found that some level of ward closure was used in 194 outbreaks, with a median closure time of 14 days and closure rate of 12.4 % . GeriatrThe literature generally suggests that ward closure is a necessary control intervention as part of a bundle –11 versuTo gain a better understanding of the role of ward closure in controlling outbreaks, we systematically reviewed the published academic literature examining the use and impact of ward closure for controlling outbreaks in the acute care hospital setting. In addition to this review, we developed a web-based environmental scan survey that was distributed to IP&C practitioners and physicians at acute care sites across Canada. The present systematic review had two objectives: (1) to identify studies that describe ward closure as an outbreak control measure in sufficient detail to determine how ward closure was defined and what was done and (2) to determine the impact of ward closure on outbreak control by answering the following question: In hospitalized patients of all ages, does the use of partial or complete hospital ward closure have a significant impact on the control of an outbreak due to invasive infection or colonization by pathogenic microbes with the potential for spread, as compared to not using hospital ward closure, with or without the use of other infection control interventions and/or practices? These two questions guided the protocol development, which then informed the screening and selection process.This review is not registered with PROSPERO.To identify relevant references for this review, we searched the following databases with no language restrictions or other limits: Ovid MEDLINE, including In-Process & Other Non-Indexed Citations, 1946 to 7 July 2014; Ovid EMBASE, 1974 to 7 July 2014; CINAHL Plus with Full Text, 1937 to 8 July 2014; and Cochrane Database of Systematic Reviews, 2005 to May 2014. Our search consisted of selected subject headings and keywords related to the use of ward closure, combined with terms for outbreaks of infectious diseases  for at least 48 h (or length not specified) as an intervention to help control outbreaks. We defined “complete ward closure” as the application of ward closure across all beds on a ward/unit and “partial ward closure” as the application of ward closure to some, but not all, of the beds on a ward/unit. “Ward closure” included any or all of the following: no new patients admitted to the area; no transfers to other units within the healthcare facility allowed unless required for ongoing care; and no transfers to other healthcare facilities, including long-term care, with no restrictions on discharge home . “Ward cThe included studies were systematically reviewed and relevant data was extracted from each article on the following parameters: study design, setting and population characteristics, causative pathogen(s), details of ward closure, details of other outbreak control interventions, outcomes relevant to the review, including the number of patients colonized and/or infected, and the role of ward closure for controlling the outbreak were extracted and recorded by one of the authors. Data from non-English full-text articles were extracted by a researcher who was a native or fluent speaker of the language and had knowledge of data extraction for systematic reviews. Relevant extracted data were collated in a descriptive summary and tabular format based on the findings from the parameters listed above.We adopted Juni and colleagues’  componenFrom the 2095 references gathered from all the sources searched, a total of 97 English and non-English articles in Dutch, French, German, Japanese, and Spanish were accepted for inclusion Fig. .Fig. 1PROf the 97 included studies, 67 were case series, 14 were case–control studies, 5 were cohort studies, 5 were before-and-after studies, 5 were interrupted time series studies, and 1 was a time series study. As there were no studies that included a controlled comparison between ward closure and other interventions, the studies included in this review only allowed us to fulfill our first objective. Thus, this review purely focused on studies that described how ward closure was used as an outbreak control intervention and its impact on the outbreak.From the details provided within the context of the setting and population, the studies were organized firstly by the organ system(s) affected and secondly by the genus of the causative pathogen within each of these organ system categories. The organ system and genus categorization lent itself very well to an additional categorization by the mode of transmission, which is the basis for infection prevention and control precautions. The organ system categories included: “gastrointestinal,” consisting of 17 studies; “respiratory,” consisting of 11 studies; and “multiple/mixed,” which includes the central nervous system, skin/soft tissue, urinary tract, eye, abdominal, and vascular or when more than one system is affected simultaneously and consisting of 63 studies. Of the studies in the third category, eight studies described predominant colonization, 12 studies described predominant infection, and 43 studies described a combination of colonization and infection. A sixth category included six studies that described the impact of infection control policies and of specific interventions on outbreak control. The modes of transmission relevant to our study included contact (both direct person-to-person and indirect via fomites and inanimate objects), droplet , and airborne .C. difficile, norovirus, rotavirus, Salmonella panama, small round structured virus, or small round structured virus and small round featureless virus. The primary mode of transmission for all these pathogens is direct person-to-person contact and indirect contact with contaminated surfaces [We identified 17 studies –37 on ousurfaces . The outThe definition of ward closure varied across the studies, and ward closure lasted between 3 days and 1 month among the studies that reported length of closure. Six studies defined ward closure as prohibiting new admissions to the affected clinical area  –26. WiddOf nine studies that reported achieving outbreak containment, six attributed it to all the measures used , 31, 33,Streptococcus pneumoniae, or Streptococcus pneumoniae and Streptococcus spp. [Eleven studies examined outbreaks of influenza A, parainfluenza, parainfluenza and respiratory syncytial virus, severe acute respiratory syndrome, cus spp. –48 The pcus spp. . The outcus spp. . In addiThe affected clinical area was closed to new admissions in six studies –44. New In the studies that achieved outbreak containment, this outcome was attributed to all the measures used in four studies , 46–48, Enterococcus, Escherichia coli, Klebsiella pneumoniae, or Staphylococcus aureus that resulted predominantly in colonization and involved 3–59 patients at single facilities [The mode of transmission for the microbes described within this category is via contact . Eight scilities –56. BetwIn six studies, no new admissions were permitted to the affected clinical area. Ward closure entailed limiting transfers and partial closure of four beds in the outbreak described by Delmare and colleagues  and restThe authors of four studies attributed outbreak containment to all the measures implemented , 52–54. Acinetobacter baumannii, adenovirus, echo 19 virus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, or Staphylococcus aureus that resulted predominantly in infection among 4–48 patients [The major mode of transmission for the microbes described within this category is via contact with the exception of adenovirus and the echovirus where both contact and droplet transmission occur . We idenpatients , 57–67. Ward closure involved closing the affected clinical area to new admissions in eight studies , 57–63. Authors reported achieving outbreak control in 11 studies. This outcome was attributed to all the measures used in three studies , 65, 67.Acinetobacter baumannii, Coxsackie virus, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, parvovirus, Salmonella, Serratia marcescens, Staphylococcus aureus, or Streptococcus. For all but one study that involved a total of seven hospitals, the outbreaks occurred at one facility and affected a total of 3–245 patients.Of the remaining studies, 43 reported outbreaks that affected other or multiple organ systems and resulted in both of infection and colonization –110. TheStaphylococcus aureus) outbreak that could not be controlled by the use of other measures, including temporary closure on three occasions [Among these studies, the definition of ward closure varied widely and lasted from 1 week to 2 months in 19 studies that reported the length of closure. Ward closure was defined as limiting and then not accepting new admissions to the affected clinical area in 30 studies –97, limiccasions . Three sccasions –110.p < 0.001) in the number of cases of colonization and infection [Successful outbreak containment was reported in the vast majority of the studies. This outcome was attributed to multiple measures in five of the studies , 104, munfection .We identified six studies that focused on the impact of specific infection prevention and control policies or a control intervention , 111–114C. difficile infection and MRSA when a new policy entailing hand hygiene, education, and restriction on antimicrobial treatment was implemented [In two studies reporting on norovirus outbreak(s), bay closures supplemented with other measures were reported to have a greater impact on the reduction of closure length and bed-days lost than ward closure as a primary intervention , 13. Altlemented . Lastly,lemented . The autOwing to the nature of the studies included in this review, a number of potential confounders and sources of bias were identified. Firstly, none of the studies controlled for confounding, and the majority of them did not address the confounding factor bias when discussing the impact of the interventions used. All of the studies used ward closure in combination with other interventions, and as such, the impact of each measure on outbreak containment could not be determined. Relatedly, there may also have been a potential for a dose–response relationship in studies that increased the extent of ward closure, for example, from closing the unit to select admissions to closing to all admissions. Secondly, there is the potential for selection bias in studies that did not use epidemiological typing and, subsequently, could not confirm that all affected patients were colonized or infected with the same strain of the causative pathogen. Another source of bias stems from the selection of specific outcomes. Furthermore, some of the case definitions relied on the presence of symptoms and did not confirm cases with any diagnostic method or epidemiological typing, resulting in case finding bias. The studies could have also been subject to recall bias as the vast majority of articles are retrospective. As all the articles included in this study reported on successfully controlled outbreaks, it is highly likely that the reviewed literature is vulnerable to publication bias. Many of the articles failed to address these potential sources of bias that may have contributed to the main findings and, particularly, the impact of ward closure on containing the outbreaks. This failure may be attributed to the retrospective and observational nature of outbreak investigation studies. Fourthly, definitions of ward closure were varied between studies, potentially creating bias in understanding the impact of ward closure and in determining whether the studies used complete or partial closure.We sought to identify studies that describe the use of ward closure as an intervention in outbreak control and determine its importance. Our systematic review expands on existing work by providing an extensive review of the epidemiological literature on the use of ward closure as an intervention to control outbreaks of pathogenic microbes among inpatients hospitalized in acute care settings. We identified 97 studies that described the use of ward closure as part of a bundled approach to their strategy. None of the studies used ward closure in a setting where it was able to be isolated as a singular control measure, limiting our assessment of the direct efficacy of ward closure on outbreak containment, which was one of our primary objectives.It was not possible to draw any firm conclusions about the impact or effect of ward closure from the studies for a number of reasons. Firstly, the use of “ward closure” varied considerably within the papers included in the review. Our review was unable to identify whether partial or complete closure was instituted in the vast majority of the studies, as precise definitions were not used to describe the type or nature of ward closure. The results suggest that there is not a universal definition of “ward closure”; rather, ward closure refers to restrictions on patient movement into and out of a unit/ward or a facility and could encompass a number of qualities and multiple phases and/or degrees of application. Secondly, with the exception of the prevention and control policy and intervention studies, all of the studies of the included papers were reports or descriptions of outbreak investigations. As investigators could not manipulate exposures , all outbreak studies were observational in nature and the results were thus susceptible to a number of potential confounders. The vast majority of the included articles did not record these potential confounders or were not adjusted accordingly in any type of additional analysis. The studies were vulnerable to multiple biases, including confounding factor bias, publication bias, and recall bias, and none of them reported taking measures to prevent them or address their source. As Cooper and colleagues  noted, tOur review highlights potential areas for further research on the role of ward closure as an intervention measure for managing and terminating outbreaks. Improving the quality of reporting can be a first step to addressing the difficulties in assessing the applicability and generalizability of these studies . Given tFurther, formal assessment of the frequency and outcomes of unit closure versus no unit closure during an outbreak could be undertaken. This should include gathering information on the type of outbreak where a unit is closed, duration of the outbreak, whether or not the unit is closed, and the impact on patient flow, examining both admission and discharge. While there are some inconsistencies in the quality and format of reporting, there are some metrics that are consistently reported, including number of beds, length of closure, and bed-days lost. This information could inform an economic study using modeling to predict the cost of implementing ward closure. Finally, there are potential ethical and legal considerations in deciding whether to implement closure of care settings during outbreaks that are not addressed in the literature reviewed nor within this review. On the one hand, failure to restrict admissions implies that new and unaffected patients are knowingly admitted to an area known to have ongoing transmission of a potential pathogen; on the other hand, closure of a clinical area may reduce access to care.While this review was undertaken with rigor and in accordance with the requirements of systematic review methodology, it is important to note its limitations. Firstly, for the majority of articles, data were extracted by a single reviewer; however, initial screening was undertaken rigorously by two reviewers, and disagreements were resolved with a third-party adjudicator. Secondly, the literature available for this review could report a positive effect of ward closure, as it is possible that there are many outbreaks that were controlled without using ward closure and were never published. Similarly, outbreaks where interventions failed to control transmission leading to endemic transmission are less likely to be published. For example, it is common for long-term care facilities to use ward (or facility) closure  to control gastrointestinal and respiratory outbreaks, and these are seldom published. While the outbreaks are generally controlled and the ward (or facility) is re-opened, the key question is whether ward closure is necessary and effective. Lastly, the review is based on the last electronic search which was completed in July 2014, and as such the review may not be entirely up to date.n = 3), geriatric wards (n = 3), and neonatal intensive care units (n = 2) were reported more than once , making up 56 % of the pathogenic species. These pathogens are known for their persistence within environmental niches and relative resistance to commonly used disinfectants.It can be concluded that ward closure for containment of outbreaks remains an intervention that is not evidence based in the traditional sense; however, this review demonstrates that ward closure is frequently used and was always used as part of a bundled approach, whether as part of a sequence of, or in parallel with, other interventions, and in this sense, is similar to other public health responses. However, it was interesting to observe that in the majority of the studies in this review, ward closure was applied in the late stages of the overall outbreak response rather than as a first measure. In addition, in 16 studies, despite the use of ward closure, additional cases continued to be reported, suggesting that ward closure was not an effective intervention in these settings. Other than general wards, which were not described well, burn units n = , geriatre Tables , and 6. There are also potential ethical considerations in the closure of wards during outbreaks that are not addressed within the context of the reviewed studies and would need to be taken into consideration by infection control personnel and hospital administrators. Admitting new and unaffected individuals to a hospital ward that is known to have ongoing transmission of a potential pathogen, particularly if associated with a high case fatality rate, warrants careful deliberation. The risk of new transmissions needs to be juxtaposed against the failure to contain the outbreak despite closure, the disruption of care delivery, and lack of access to care for other patients and overloading other care units, particularly emergency departments, where the risks of overcrowding and delayed care present other challenges.With no published controlled studies associated with a benefit from ward closure, infection control practitioners and hospital administrators will need to continue to balance the competing risks, taking into consideration the nature of the outbreak, the type of pathogen and its virulence, mode of transmission, and the setting in which it occurs and take reasonable steps to protect patients, and since ward closure has been used in the past, it will likely continue to be used as an intervention strategy until better quality evidence is available.The present systematic review could not ascertain the impact of ward closure on outbreak containment for any of the included studies based on our primary objective. Ward closure was commonly reported as an intervention during the course of a wide range of outbreaks, and outbreak control was described in most settings with the use of ward closure, usually in the late stages of the outbreak and was always used in parallel or in sequence with other interventions. Our results highlight that there is no universal definition of ward closure, as it has been defined in various and imprecise ways in the included studies. Since the published literature to date consists of uncontrolled observational study designs that were vulnerable to a number of potential confounders and biases, the actual impact of ward closure could not be determined. Our review has identified a number of research gaps and new opportunities for future investigations. In particular, the ability to determine the generalizability and applicability of ward closure as a control intervention could be improved by standardizing outbreak investigation reporting to include information on the use, role, precision of definition, and timing of ward closure.