Tissue clearing methods promise to provide exquisite three-dimensional imaging information; however, there is a need for simplified methods for lower resource settings and for non-fluorescence based phenotyping to enable light microscopic imaging modalities. Here we describe the simplified CLARITY method (SCM) for tissue clearing that preserves epitopes of interest. We imaged the resulting tissues using light sheet microscopy to generate rapid 3D reconstructions of entire tissues and organs. In addition, to enable clearing and 3D tissue imaging with light microscopy methods, we developed a colorimetric, non-fluorescent method for specifically labeling cleared tissues based on horseradish peroxidase conversion of diaminobenzidine to a colored insoluble product. The methods we describe here are portable and can be accomplished at low cost, and can allow light microscopic imaging of cleared tissues, thus enabling tissue clearing and imaging in a wide variety of settings. The ability to phenotype tissues in three dimensions is important for both clinical and research settings; the development of tissue clearing methods allows phenotyping of intact tissues with high resolution2344A second limitation of most clearing methods is the use of fluorescently labeled molecules and probes to generate specific labeling and contrast8Here we describe a more portable implementation of tissue clearing, based on PACT, termed SCM (simplified CLARITY method). We show that polymerization and clearing can be performed with minimal equipment. Furthermore, we show that we can utilize colorimetric methods for specific labeling and imaging of tissues based on the diaminobenzidine reaction and the resulting tissues can be imaged with visible light microscopy techniques. These methods hold promise to enable tissue processing, clearing, and imaging at reduced cost and to enable additional, complementary microscopic methods to be utilized to image the resulting tissues.The simplified CLARITY method (SCM) generates tissue-hydrogel hybrids with acrylamide by increasing the concentration of the initiator for the polymerization reaction, thus eliminating the need for vacuum purging and nitrogen backfilling. Solutions of initiator must be freshly made as we have found that polymerization efficiency decreased with increasing age of the initiator, likely due to oxidation. It is critical to ensure complete washing of samples with multiple phosphate buffered saline rinses (or other buffered aqueous solutions) prior to cross-linking with paraformaldehyde, in order to thoroughly remove red blood cells before they lyse. Red blood cell accumulation and lysis in samples may lead to hemoglobin polymerization, which contributes to autofluorescence. Although a method to decolorize heme using aminoalcohol has been described2The SCM-generated tissues preserve endogenous genetic fluorescence (if present) and are compatible with most immunohistochemical techniques including labeling with specific antibodies. For fluorescently labeled specimens, we utilized light sheet microscopy (LSM) to image fluorescent labels within intact cleared tissues, to enable the deepest tissue imaging possible at a fast rate, as we have previously shown that LSM offers the greatest depth of view and most rapid image acquisition as compared to commercially available confocal microscopy systems1113Almost all tissue clearing methods have been developed to enable fluorescence imaging. While useful in many cases, this excludes the use of light microscopic techniques such as phase microscopy which can provide complementary information about tissue samples. In addition, many of the portable microscopy solutions such as those utilizing holographic lens-free microscopy or cell phone cameras with additional attachments, are based on light microscopy. To allow cleared tissues to be imaged with additional non-fluorescence based modalities, we thus wished to determine parameters to enable color-based contrast imaging using colorimetric labeling of SCM processed tissues. As a proof of concept, we labeled parvalbumin neurons in slices of adult mouse cortex. The parvalbumin neurons are a unique subset of neurons that are few in number and help to synchronize cortical signals; they can be labeled specifically using anti-parvalbumin primary antibodies. Instead of utilizing a fluorescently-labeled secondary antibody, we utilized secondary antibodies labeled with horseradish peroxidase. On exposure to the substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB), the horseradish peroxidase converts the DAB to an insoluble brown product of micron-sized particles that can be visualized with epi-illumination visible light microscopy or phase microscopyTo determine the correct parameters to enable DAB staining, we utilized 50 and 100 μm sections of adult mouse brain tissues. We determined that for these 50 μm and 100 μm sections, the secondary-labeled tissue should be incubated with DAB for 2–6 minutes for maximum tissue contrast and minimal background staining, at the concentration of DAB solution we utilized. Longer DAB incubation times may cause non-specific staining or diffusion of the particles away from the reaction site. The exact incubation time depends on the thickness of the tissue and the concentration of antibodies utilized. We then imaged the resulting DAB-stained tissues using epi-illumination light microscopy and determined that we could specifically visualize parvalbumin neuronal bodies and some of the dendritic and axonal processes. Tissue clearing offers an unprecedented ability to image critical structures and features in three dimensions and offers a complementary approach to traditional histopathological analysis using formalin-fixed and paraffin-embedded tissue sections. The methods we have described are a significant improvement and simplification over currently available techniques and will allow for tissue clearing to be utilized in almost any setting due to its independence from bulky processing equipment. The main differences of SCM to previously published protocols involve the reduction in clearing reagent use and the simplification of the protocol to eliminate the need for reducing oxygen tension in the sample and thus the need for vacuum pumps or nitrogen gas sources while maintaining sample fidelity and integrity and comparable clearing times. The resulting cleared tissues maintain endogenous expression of genetically-encoded fluorescent proteins, which are compatible with a broad variety of antibody-labeling approaches, and most importantly now have the ability to be imaged with light microscopy using the colorimetric DAB stain approach as first reported here. The resulting images appear similar to light microscopic histological images, but also allow intact deeper-tissue imaging. The use of colorimetric stains allows tissues to be analyzed using light microscopy and phase contrast microscopy and potentially with portable microscopic approaches. We did not observe appreciable diffusion of the DAB particles in the tissue, thus demonstrating the stain specifically localizes to and labels features of interest. In addition, other color-based contrast methods can also be incorporated to allow multiplexed colorimetric phenotyping with high resolution.Tissues cleared with SCM can be visualized with a variety of microscopic systems. Tissues with genetic fluorescent proteins or labeled with fluorescent markers can be imaged with several modalities, including epifluorescence, confocal, or light sheet microscopy. We have primarily utilized light sheet microscopy to enable rapid imaging of thick tissues; however, confocal or epifluorescence microscopy can also yield structural and phenotypic information in an appropriately labeled sample18SCM and the colorimetric contrast-generating methods can allow tissue clearing approaches to have a wider reach. The ability to utilize phase contrast and other non-fluorescence based microscopic approaches can increase the information that can be gained from such tissues. We envision that these methods can also be utilized in research laboratories in lower resource settings, as they are simple to perform and do not require complex or costly equipment.Mouse tissues were harvested according to the UCLA IACUC guidelines and all experimental protocols were approved by the IACUC. Briefly, mice were deeply anesthetized with pentobarbital and perfused transcardially with saline followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). Human skin cancer tissue samples were obtained from patients undergoing excision of their cancers after appropriate informed consent and under approval of UCLA IRB#12–01195, in accordance with UCLA IRB guidelines. Tissue samples were obtained from sections of tumors not necessary for diagnostic or margin control purposes and varied in size depending on the size of the original skin cancer. All tissues were rinsed in PBS three times for ten minutes and then placed in 4% paraformaldehyde (Electron Microscopy Sciences) at 4 °C overnight. Brain tissues to be sectioned were then embedded in 3% Bactoagar and free-floating coronal sections  were collected using a vibratome (Leica Microsystems).The tissues were then placed in a 4% acrylamide solution (Bio-Rad) along with 0.5% w/v of the photoinitiator 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride ; this is double the concentration of the original PACT CLARITY protocol. The tissues were then incubated overnight at 4 °C followed by incubation at 37 °C for 2–3 hours to initiate polymerization of the acrylamide. After polymerization, the tissues were rinsed with PBS and then placed into a clearing solution comprised of 8% w/v sodium dodecyl sulfate  and 1.25% w/v boric acid (Fisher) (pH 8.5). The samples were incubated at 37 °C until cleared. Tissues were then rinsed for one day in 1X PBS after clearing to remove residual SDS.To prepare cleared mouse brain tissues for the DAB stain of parvalbumin-containing neurons (Pab), rat monoclonal anti-mouse Pab primary antibodies (ImmunoStar) were diluted in a 1:200 volume/volume ratio with blocking solution, which was comprised of 1% v/v goat serum and 0.1% v/v Tween-20 in PBS (hereafter referred to as goat blocking solution).The cleared mouse brain sections were incubated in 200 μL of the primary antibody-blocking solution mixture for 48 hours shaking at room temperature. Subsequently, the cleared brain sections were washed with 1x PBS for 24 hours with rotation at room temperature. For fluorescent labeling, goat monoclonal anti-rat antibodies conjugated with Alexa-594 (Pierce Antibodies) were diluted in a 1:200 v/v ratio with goat blocking solution to form the secondary antibody-blocking solution mixture. For colorimetric imaging, goat monoclonal anti-rat antibodies conjugated with horseradish peroxidase enzyme (Thermo Scientific Pierce Antibodies) were diluted in a 1:200 volume/volume ratio with goat blocking solution. The cleared mouse brain sections were incubated with the appropriate secondary antibody-blocking solution mixture for 48 hours with rotation at room temperature. Afterwards, the cleared mouse brain sections were washed with 1x PBS for 24 hours with rotation at room temperature.To prepare the DAB solution for colorimetric imaging, a 3,3′-Diaminobenzidine (DAB) solution was formulated with 250 μL of 1% w/v 3,3′-Diaminobenzidine (Sigma), 250 μL of 0.3% hydrogen peroxide solution (Sigma), and 4.5 mL of DI water. The solution was then adjusted to a pH of 7.4 with 1N sodium hydroxide .The cleared mouse brain sections labeled with the horseradish peroxidase secondary antibody were placed in the DAB solution for 2 minutes, 4 minutes, and 6 minutes respectively to form light, medium, and dark stains. These samples were then washed with 1x PBS to remove the excess DAB solution. The DAB stained samples were placed in refractive index matching solution (RIMS) for imaging and storage. The RIMS formulation is as follows: to make 30 mL RIMS, dissolve 40 grams of Histodenz (Sigma) in 0.02 M Phosphate buffer (Sigma) with 0.05% w/v sodium azide (Sigma) and syringe filtered through a 0.2 μm filter. The solution can be stored at room temperature.f = 50 mm, LJ1695RM-A, Thorlabs Inc, New Jersey, USA) and was then reshaped by a group of achromatic doublets . After passing through an f = 150 mm lens, the beam expanded to a sheet with the width of 40 mm, as well as the waist of 17 μm. The detection module was installed perpendicular to the illumination plane, and it was composed of a stereo microscope  with a 1x magnification objective , a scientific CMOS  and optical filters . The sample with RIMS and 1% agarose solution was embedded in a borosilicate glass tubing with an inner diameter of less than 6 mm, and then the mounted sample was placed on a motorized translational stage. Both the sample and its holder, but not the translational stage, were immersed in a transparent chamber filled with 99.5% glycerol to match the refractive index among different materials. Illumination and detection were controlled by a computer with dedicated SSD RAID 0 storage for fast data streaming.Light sheet imaging was carried out on a home-made system . A diodeColorimetrically labeled tissues were imaged using a Zeiss Axiovert inverted microscope with the included epi-illumination light source. Brain sections labeled with Alexa 594-labeled secondary antibodies were imaged using this Zeiss Axiovert microscope with a xenon-mercury arc lamp and appropriate excitation and emission filters. Images were collected using a Photometrics CoolSnap HQ2 camera.How to cite this article: Sung, K. et al. Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping. Sci. Rep.6, 30736; doi: 10.1038/srep30736 (2016).


Neutrophils are highly abundant innate immune cells that contribute to asphyxic episodes of acute asthma exacerbations, and secrete the proinflammatory cytokine tumour necrosis factor-α (TNF). Recycling endosomes (REs) are specialized secretory compartments that perform multiple functions including trafficking of cytokines to cell surfaces, although these are not characterized in neutrophils. Our objective is to identify trafficking components in neutrophils that may contribute to cytokine secretion.The effect of bacterial lipopolysaccharide (LPS) stimulation on the trafficking of stored and newly synthesized TNF was determined by treatment of human peripheral blood neutrophils with or without cycloheximide. To visualize intracellular TNF, neutrophils were adhered to glass slides and treated with LPS for 1 h (10 ng/ml). Colocalization of TNFα in neutrophils was performed with transferrin-Alexa 488 and anti-VAMP3 (markers for RE), anti-CD63 and anti-CD66b a membrane markers for the primary and secondary granules in neutrophils, respectively. We also determined Rab5 and Rab7 colocalization with TNF . Imaging was carried out by Deltavision OMX super resolution microscopy.LPS induced 30-40% TNF secretion from stored sources, with the remainder newly synthesized. We found that neutrophils possess REs as determined by transferrin uptake and VAMP3 labeling. TNF also colocalized with REs, primary and secondary granules as well as early and late endosomes, suggesting multiple sites of TNF storage and trafficking in neutrophils. However, TNF only colocalized with VAMP3 around periphery of cells after 1 h stimulation with LPS, suggesting TLR4-induced TNF trafficking via REs.+VAMP-3+ vesicles towards the cell periphery in response to LPS. This suggests that neutrophils utilize REs for trafficking of TNF to the cell surface in response to TLR4 signalling. These findings contribute to our understanding of how neutrophils package, transport, and release cytokines.The present study provides evidence that movement of TNF


Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. colicommon pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract.Uropathogenic  Escherichia coli strains of fecal origin are able to ascend the urethra and colonize the bladder through recognition of uroplakin, a mannose-rich proteinaceous plaque found at the luminal surface of bladder epithelial cells. Pili-mediated bacterial attachment triggers a myriad of host-initiated processes including cytokine production, apoptosis and exfoliation E. coli (UPEC) into bladder epithelial cells is mediated by the FimH adhesin of the type 1 pilus (T1P). UPEC invasion involves fusiform vesicles, cyclic AMP, Toll-like receptor 4 (TLR4) and integrins Bacterial adherence is the first step in host tissue colonization and development of disease and it is generally a multi-factorial event that involves the participation of fimbrial and non-fimbrial adhesins orchestrated, in turn, by a myriad of regulatory elements influenced by environmental and host signals at different stages during the infectious process Several pili types, including T1P, pyelonephritis-associated pilus (Pap) and S pili, and the Dr adhesins of UPEC contribute to infections in the urinary tract E. coli (MENEC) strains produce a fimbrial structure called “meningitis-associated temperature-dependent fimbriae” or Mat upon growth in Luria-Bertani broth at 26°C E. coli; however, it was later shown that the production of Mat fimbriae was not restricted to MENEC strains or to growth at temperatures below 37°C E. coli strains and that the structural pilin gene matB is widely distributed and highly conserved amongst E. coli strains. For these reasons we proposed to call these fimbriae “E. coli common pilus” or ECP E. coli (ETEC) to cultured epithelial cells, suggesting that ECP may function as intestinal adherence factor for these E. coliecp operon exists in Klebsiella pneumoniae, an organism also capable of causing UTIs, and nearly 100% of clinical strains tested produced ECP, in association with the MR/K fimbriae, when adhering to cultured human epithelial cells E. coli and Klebsiella, the aim of this study was to investigate the contribution of ECP to the adherence and invasion capabilities of UPEC.Clinical meningitis-producing Animal care use was reviewed and approved by the University of Florida IACUC.Bacterial strains and plasmids used in this study are listed in For the various immunoassays performed, we used highly absorbed rabbit antibodies specific for ECP, which were available from previous studies 2 atmosphere. For adherence and invasion assays, cell monolayers were propagated in the appropriate culture medium until 80% confluent, washed with phosphate buffer saline  and transferred to 24-well plates containing DMEM with 0.5% D-mannose and with or without glass coverslips Human HeLa (cervix) and HTB-4 (bladder)  epithelial cells were maintained in DMEM with conventional antibiotics and 5% fetal bovine serum at 37°C in an incubator with 5% CO7 bacteria) of a DMEM overnight culture of the indicated strains. To avoid cytotoxicity and cell lysis observed after prolonged incubation periods of cultured epithelial cells with UPEC strains, adherence assays were carried out for 2 h as previously described HeLa and HTB-4 cells were infected with 10 µL  and goat anti-rabbit IgG conjugated to Alexa-Fluor 488 (1∶100) (Molecular Probes). Cellular and bacterial DNA was stained briefly with Hoechst nucleic acid stain (Molecular Probes), washed with PBS, mounted on glass slides, and then viewed in an Axio Imager1.0 Zeiss microscope (Zeiss). Replica samples were washed and viable bacteria, in the absence or presence of gentamycin, were enumerated by plating ten-fold serial dilutions onto LB agar containing the appropriate antibiotic. Statistical significance of these results was determined using the unpaired parametric Student's t-test.Even size sections (2 mm × 2 mm) of murine bladder transitional epithelium (urothelium) were infected in triplicate ecpA following the procedure reported by Datsenko and Wanner ecpA sequences flanking a kanamycin cassette, using DNA of plasmids pKD4 as template. This fragment was electroporated into competent UPEC strains carrying plasmid pKD46 encoding the lambda Red recombinase, which expression was induced by adding L-(+)-arabinose (Sigma) at a final concentration of 10 mM. The mutation was confirmed by PCR using primers flanking ecpA (G90 and G91), as well as primers inside the kanamycin cassette (K1 and K2), and chromosomal DNA from Km resistant Ap sensitive colonies as template. The loss of ECP in the mutants was assessed by several immunoassays using antibodies against ECP. The ecpA mutants were complemented with ecpAB on plasmid pMAT9 to restore ECP production ecp genes from plasmid pMAT9 were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Promega).UPEC strains F11 and CFT073 were targeted for mutagenesis of 600. Equal numbers of bacteria were used to prepare whole-cell extracts after treatment with acidified water (pH 1.2), boiling for 5 min, addition of SDS-PAGE sample buffer and neutralization with 1N NaOH, as previously described Overnight bacterial cultures obtained from DMEM were adjusted to an absorbance of 1.1 at ODBacterial cultures were spotted onto 300-mesh carbon-Formvar copper grids, negatively stained with 10 µL of 1% phosphotungstic acid (pH 7.4) for 5 min, and analyzed for the presence of pili by transmission electron microscopy (TEM). Immuno-electron microscopy (IEM) studies were performed to confirm the presence of ECP by incubating the bacteria for 1 h with rabbit anti-ECP antibody (diluted 1∶10) in PBS containing 10% BSA and 1 h incubation with goat anti-rabbit IgG conjugated to 10-nm gold particles diluted 1∶10 , as previously described E. coli strains were tested for biofilm formation onto glass coverslips in 24-well plates (Cellstar). Fifty μL of bacteria were added to each well containing 1 ml of DMEM supplemented with 0.5% D-mannose and incubated for 24 h at 37°C. The biofilms were fixed with 2% formaldehyde, stained with a solution of Giemsa and visualized under light microscope. For quantitative determinations, absorption of Crystal Violet dye by biofilms was measured at 595 nm with methanol as previously described fliC negative control, all other strains showed motility.CFT073 and derivative strains were spiked on 0.3 % motility LB agar. After overnight incubation at 37°C, the halos obtained represented bacteria capable of swimming. Except for the ΔE. coli isolates collected from January 2010 to December 2011 were obtained from midstream urine samples of women clinically diagnosed with acute UTI from the Urology Service at the General Hospital "Dr. Manuel Gea González" in Mexico City. Bacteria included in this work were from cultures with counts >105 CFU/mL or more. Isolates were identified by MicroScan 4 walkaway (Dade Behring). Pure cultures were maintained at −70°C in Brain-Heart infusion broth/glycerol 50%. Genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen) according to manufacturer's instructions. PCR amplifications were performed using a TopTaq master mix kit (Qiagen). For ecpA amplification, a set of primers (5′-TGAAAAAAAAGGTTCTGGCAATAGC-3′) and (5′-CGCTGATGAGGAGAAAGTGAA-3′) were used for the initial PCR. A second set of primers (5′-GTGACATGGCAAAATGATTACAGC-3′) and (5′-TCACGGGAATGAACTTATCACCC-3′), were used for ecpRB amplification to search for a possible modified form of ecpA in PCR-negative strains. UPEC strain CFT073 was used as a positive control.Three mL of midstream urine from de-identified patients with acute UTI were centrifuged at 3000 rpm for 30 min at 4°C and the cell pellet was washed twice with PBS, placed in glass coverslips, and fixed with 3% paraformaldehyde for 15 min. An urine sample from a clinically healthy woman was used as a negative control. The preparations were PBS-washed two times and blocked with PBS-10% horse serum for 20 min at room temperature. A 1∶1500 dilution of the primary anti-EcpA antibody was added to the samples and incubated overnight at 4°C. After washing, secondary anti-rabbit IgG antibodies  were incubated for 40 min. The epithelial cells were stained for 1 h at room temperature with phalloidin-rhodamine diluted 1∶120 and washed two times with PBS. The preparations were counterstained with VectaShield (Vector Laboratories) that contains DAPI, and mounted on glass slides. Microscopical analysis was done with a Leica DM100 immunofluorescence microscope (Leica Microsystems) and microphotographs were taken with an Olympus FV100 confocal microscope (Olympus America Corporate).E. coli strains isolated from women with acute UTI with ages ranging between 12–44 years (mean age of 33.9 years) attending the General Hospital Dr. Manuel Gea González in Mexico City, were analyzed for the presence of ecpA by PCR. One hundred and seventy (98.83%) isolates were positive for ecpA and the 2 ecpA-negative strains were also negative for ecpR and ecpB genes flanking ecpA, indicating that some strains possess the ecpA gene with genetic variations or have lost ecpA. These data show that the majority of UPEC strains contain the ecpA gene.A total of 172 e. g. >30,000X) under the electron microscope  flexible fibers that protruded from the periphery of the bacterial surface when observed at high magnifications  and ecpB (chaperone pilus biogenesis gene) ecpA mutants in immunoblots  in adherence was observed for both mutants to both cell lines. CFT073 ΔecpA manifested 52% reduction in adherence to HeLa cells and was 3 times less adherent to HTB-4 bladder epithelial cells than the wild-type strain (ecpA mutant was confirmed by light microscopy observation (ecpA was reduced by 57% in adherence to HeLa cells (data not shown). The residual adherence in the mutants is not surprising considering that these bacteria possess numerous adhesive systems. Complementation of the ecpA mutants with ecpAB in plasmid pMAT9 restored adherence to wild-type levels in both strains  to these cell lines suggesting that ECP presumably also plays a role in the invasive properties of this E. coli. Restoration of ECP production in the mutant carrying plasmid pMAT9 recovered the invasive properties of this strain . Since 1% D-mannose inhibited cell adherence and yeast agglutination we conclude that T1P levels produced are not affected in any of the strains either in the presence of epithelial cells or yeasts.T1P have been reportedly associated with the pathogenesis of UTI by UPEC strains and thus we were interested in knowing the contribution of these pili to the adherence of CFT073. To do this, we performed an adherence assay in the presence of 0, 0.5 and 1% D-mannose, which is known to inhibit T1P-mediated adherence. Adherence of CFT073, the f CFT073 . We alsoin vitro observations with cultured epithelial cells regarding production of ECP and adherence could be reproduced when infecting mice bladder urothelium ex vivo. In addition, we were interested in assessing and comparing the invasive capabilities of the CFT073 wild-type strain versus the ecpA mutant. To this purpose, we also used the gentamycin protection assay and sectioned the infected bladder tissues to visualize the bacteria and ECP production. We found that the CFT073 ecpA mutant was slightly deficient in adherence compared to the wild-type strain but did not reach significance (ecpA mutant was strongly reduced (p = 0.0001), providing compelling evidence for the first time that ECP mediates invasion of UPEC  produce ECP  displayed fluorescent pili  D-mannose significantly reduced T1P-mediated adherence of the wild-type and derivative isogenic strains. Thus, the data indicate that ECP works in synchronicity with other adhesins presumably to ensure host cell colonization.In this study, we show that the newly described ECP is yet another key player in the general scheme of the interaction of UPEC with host cells. The basis for this assumption comes from compelling IEM, IFM, and molecular data indicating that UPEC produces ECP when incubated with cultured epithelial cells and during biofilm formation on a glass surface. Most interestingly, ECP production was manifested with specific anti-ECP antibodies in the mouse bladder urothelium E. coli pathogroups E. coli in the mouse bladder urothelium and exfoliated bladder epithelial cells present in urine sediments from an UTI patient are intriguing and highly evocative of a role for ECP in the biology of this organism within the urinary tract of the host. The ecpA mutant had a drastic defect in invasion of HeLa and HTB-4 cells and the murine uroepithelium. There are numerous fibrillar structures that are associated with intracellular development in UPEC Our data provokingly suggest that production of ECP may be necessary for stabilizing the extracellular bacteria adhering to the host cell membrane favoring tissue colonization and perhaps in intracellular biofilms. Other studies have shown that ECP is important for bacteria-to-bacteria interactions during biofilm formation of other E. coli has the ability to colonize many different niches within a host. This is mainly possible due to an assortment of cooperative and some times seemingly redundant adhesive structures. In this context, UPEC ECP may act only as an auxiliary adhesin at a particular stage of the infection in the human urinary tract, thus it remains to be determined whether ECP contributes to human UTI. Since a vast majority of the UPEC bacteria exit the urinary tract and then the host through urination after escape from the intracellular residence, it is possible also that ECP contributes to binding in the next environment that UPEC encounters after leaving the host. UPEC strains are commensal organisms in the gastrointestinal tract. The reported contribution of ECP in the binding of EHEC O157:H7 and fecal E. coli to intestinal epithelial cells in vitro, reasonably suggests that ECP could also be important for UPEC to establish a niche in the gut. The presence of ECP in the majority of E. coli strains ex vivo) and in exfoliated cells present in the urine of UTI patients. The observation that ECP is common to most E. coli strains, including normal flora as well as human and pathogenic E. coli strains, suggests that it may not have a specialized role in any particular disease state, but contributes to the normal lifestyle that all E. coli strains share. Although intracellular residence is not a common lifestyle amongst all E. coli strains, living in a host certainly is.In summary, this study shows that UPEC strains are capable of producing ECP under biologically relevant conditions and highlights a role for ECP as an accessory adherence factor, which in conjunction with other adhesins, may contribute to the multi-factorial complex interaction of UPEC with host uroepithelial cells.


Exome sequencing has become more and more affordable and the technique has emerged as an important diagnostic tool for monogenic disorders at early stages of investigations, in particular when clinical information is limited or unspecific as well as in cases of genetic heterogeneity.We identified a consanguineous Pakistani family segregating an autosomal recessive phenotype characterized by muscular hypertrophy, mild mental retardation and skeletal abnormalities. The available clinical information was incomplete and we applied whole exome sequencing in an affected family member for the identification of candidate gene variants.BSCL2 gene (c.574-2A > G). Expression analysis revealed that the mutation was associated with skipping of exon 6. BSCL2 mutations are associated with Berardinelli-Seip congenital lipodystrophy and a clinical re-evaluation of affected individuals confirmed the diagnosis.Exome sequencing identified a previously unreported homozygous mutation in the acceptor splice site of intron 5 in the BSCL2 gene suggesting congenital generalized lipodystrophy. Incomplete phenotypic delineations are frequent limiting factors in search for a diagnosis and may lead to inappropriate care and follow-up. Our study exemplifies exome sequencing as a powerful diagnostic tool in Mendelian disorders that may complement missing clinical information and accelerate clinical diagnosis.Exome sequencing is a powerful technique for the identification of candidate gene variants in Mendelian traits. We applied this technique on a single individual affected by a likely autosomal recessive disorder without access to complete clinical details. A homozygous and truncating mutation was identified in the  Mutation detection in genetic diseases is important for a definite diagnosis for genetic counseling, future prenatal diagnosis and therapy. Next generation sequencing technologies are increasingly being used as a diagnostic tool for genetic disorders and have been shown particularly successful in genetically heterogeneous phenotypes, or when ascertainment of clinical features and symptoms are unattainable -10. FurtBSCL2 gene previously associated to congenital generalized lipodystrophy type 2 (CGL2) [We report herein on a family segregating short and muscular stature, mild mental retardation and acromegalic appearance as an autosomal recessive trait. The clinical information was otherwise sparse. Using whole exome sequencing (WES), we identified a homozygous mutation in the acceptor splice site of intron 5 of the 2 (CGL2) . FunctioWe identified a consanguineous six generation Pakistani pedigree with three members, two females and one male, who presented with similar features. The pedigree structure suggested an autosomal recessive inheritance Figure A. The faBSCL2 variant was confirmed by direct sequencing with dideoxy chain-termination method (Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit) on a 3730xl DNA Analyzer . Sequence analysis was performed using the Sequencer software .DNA from an affected individual (V3) was sonicated using a Covaris S2 instrument . Fragment libraries were created from the sheared samples using the AB Library Builder System  and target enrichment was performed using the Agilent SureSelect Human All Exon v4 kit according to the manufacturer's protocols . Exome capture was conducted by hybridizing the DNA libraries with biotinylated RNA baits for 24 h followed by extraction using streptavidin coated magnetic beads. Captured DNA was then amplified followed by emulsion PCR using the EZ Bead System (Life Technologies) and sequenced on the SOLiD5500xl system, generating over 100 million reads of 75 bp length for each of the samples. Alignment of reads to the human reference sequence (hg19 assembly) and variant detection was performed using v2.1 of the LifeScope Software (Life Technologies). SNPs and indel data was stored in an in-house exome database together with variant annotation information obtained from ANNOVAR  and dbSNBSCL2 e5F (GTG ATG CTG CAT TAC CGC TCA GAC) and BSCL2 e7R (CTG CAA AGA GAA GCG GTG TCG G). The PCR product was re-amplified using primers BSCL2 e5F Hind III (AGC TCA AGC TTC GTG ATG CTG CAT TAC CGC TCA GAC) and BSCL2 e7R Kpn I (CCC GCG GTA CCG CTG CAA AGA GAA GCG GTG TCG G) to introduce restriction sites. The resulting PCR product was cloned into a TA cloning vector  and individual clones were analyzed by Sanger sequencing. Clones representing the two alleles  and mutant allele (gg)) were identified and subsequently inserted into a pEGFP-C2 expression vector  using Hind III, Kpn I and Rapid DNA ligation kit  and confirmed by sequencing. Thus, the minigene was fused to green fluorescent protein  was used for amplification with primers  was used2 in a humidified atmosphere. Expression of the GFP-minigene was confirmed by fluorescence microscopy after 48 hours. Subsequently, cells were harvested and total RNA was isolated using TRIZOLTM (Invitrogen), following the manufacturer’s protocols. Splicing of the minigene was detected by reverse transcription of 2 μg of total RNA using oligo-d(T) primers, utilizing the VILO cDNA synthesis kit (Invitrogen). Splicing products were amplified by standard PCR with primers Assay Forward (TAC AAG TCC GGC CGG ACT CAG ATC) and BSCL2 e7R . Cells were grown in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 20 IU penicillin/streptomycin and non-essential amino acids  at 37°C with 5% COBSCL2 gene  . Segregation of a homozygous region surrounding the BSCL2 variant was confirmed using microsatellite markers  and, indeed, clinical re-evaluation of our patients confirmed characteristic clinical features of the disease in all three affected members [BSCL2 and the clinical overlap with the preliminary information we had of the family, this was considered the most likely candidate variant of the ones indentified. Furthermore, we could by using a mini-gene construct spanning exons 5–7 of the mutated BSCL2 allele show that the mutation results in skipping of exon 6, an altered reading frame and a predicted early termination of the protein. Individuals with CGL2 can usually be recognized at birth, or soon thereafter, due to a near-total lack of body fat and prominent muscularity. Later characteristics include insulin resistance, hypertriglyceridemia, hepatic steatosis and early onset of diabetes. A high prevalence of mild to moderate intellectual impairment has been observed and patients have a higher risk for premature mortality, mainly due to cardiac failure. Additional features include acanthosis nigricans, hepatomegaly, hyperandrogenism and skeletal muscular hypertrophy [We identified a consanguineous family segregating autosomal recessive muscular hypertrophy mild mental retardation and acromegaloid appearance in three individuals. Detailed clinical and biochemical data were unavailable and we decided to perform WES on a single affected family members in search for homozygous and potentially damaging gene variants. Analysis of WES data showed a homozygous acceptor splice site mutation, c.574-2A > G, in intron 5 of the  members . Taken tertrophy ,14. The ertrophy ,15-18. Certrophy .In our family, the initial information suggested a disabling condition segregating as an autosomal recessive trait. The clinical data were insufficient for a diagnosis and further investigations of the family were impossible within a reasonable time-frame. Thus, we decided to apply WES and, from our findings, we were able to suggest that the patients were affected by CGL2 within a few weeks from the sampling of DNA. Much later, the diagnosis was confirmed through directed clinical and biochemical investigations of the family. Moreover, this exemplifies a time-saving aspect of exome sequencing in situations of diagnostic challenges, inaccessible clinical data as well as acute illnesses . Furthers BSCL2 mutation in a family segregating an autosomal recessive trait. The mutation was analyzed in a cell-based assay that confirmed exon skipping. Using the findings from WES we could suggest the diagnosis of CGL2 in advance of complete clinical information from the patients. The use of exome sequencing accelerated and facilitated the subsequent and confirming investigations, with implications for care and follow-up of affected individuals.Taken together, our results show the usefulness of WES for the identification of a single homozygouThis study illustrates how whole exome sequencing (WES) can be used in a clinical setting to identify gene mutations in search for a specific diagnosis. We show that WES can be used to accelerate clinical investigations of heterogeneous Mendelian traits for yet inconclusive cases.BSCL: Berardinelli-Seip congenital lipodystrophy; CGL: congenital generalized lipodystrophy.The authors declare that they have no competing interests.JS helped in the design of the study, carried out the functional analyses and helped to draft the manuscript. TNK, MT, PA and KM carried out the molecular genetic studies. SMB and TNK carried out the clinical evaluation of the patients. JK designed the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/15/71/prepubA) Minigene construct consisting of exon 5 through to exon 7 of the BSCL2 gene, including the introns, fused to green fluorescent protein (GFP). Two minigenes representing wt (ag) and mutated (gg) splice site were used. B) PCR based assay of the minigenes. An observed band of expected size in the wt construct (420 bp), but a smaller product (322 bp) corresponding to the expected exon skipping could be identified from the mutated construct. C) Sanger sequencing of cDNA from mutated (gg) construct confirms skipping of exon 6.Click here for fileHomozygous variants identified by WES. *snp137 indicates if the variant is present in the 137 version of dbSNP. Note that presented variants, besides the identified variant in the BSCL2 gene, are not confirmed by Sanger sequencing, and could thereby possibly be sequencing errors.Click here for file


Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins.The eastern North American monarch butterfly,  Danaus plexippus) long-distance migration from its northern breeding range in southeastern Canada and northeastern USA to its overwintering roosting sites in central Mexico , which encodes the main transcriptional repressor of the CLOCK (CLK):CYCLE (CYC) activation complex within the migration-relevant clock mechanism . Eggs were transferred to petri dishes containing milkweed leaves until hatched. Larvae were fed milkweed up to their second instar and then transferred onto individual cups of semiartificial diet. Larvae were raised in Percival incubators at 25°, 70% humidity, and under 15 hr light, 9 hr dark  conditions. Adult monarchs were housed in glassine envelopes in the same lighting and temperature conditions, and were manually fed a 25% honey solution daily.Monarch butterflies were reared in the laboratory as previously described  were utilized for base recognition in the remaining sequence positions. The TALE monomers comprised 16.5–19.5 modules with a 16 bp gap separating the TALEN target sites. TALE module arrays were constructed using a modified Voytas golden gate system (TALEN target sites were selected within the 5′-end of the cry2 and clk coding sequences using the Jack Lin’s CRISPR/Cas9 gRNA finder (http://spot.colorado.edu/~slin/cas9.html). The cry2 gRNA target site was chosen to overlap with the previously described cry2 ZFN target site (BsaI cleavage sites.The gRNA target sites were selected within the 5′-end of the In vitro transcription of TALEN mRNAs was performed using the mMessage mMachine T7 kit (Ambion) using pJDS-TALEN constructs linearized with PmeI and phenol-chloroform purified, and were polyadenylated using the Poly(A) Tailing Kit (Ambion), according to the manufacturer’s instructions. In vitro transcription of Streptococcus pyogenes Cas9 mRNA was performed using the mMessage mMachine T3 transcription kit (Ambion), using the pCS2-nCas9n expression plasmid from Addgene  according to the manufacturer’s instructions. RNAs were quantitated by spectrophotometry (NanoDrop 1000) and diluted in RNase-free water to a final concentration of 0.5 μg/μl for cry2 TALENs and clk TALENs, 0.5 μg/μl for Cas9, and of 0.1 μg/μl for cry2 and clk sgRNAs.To perform microinjections, eggs were collected as previously described  competent cells induced with IPTG and purified, as previously described (For cry2 and clk primers (given in Table S1). PCR products were purified using 1.5 × modified Sera-Mag Magnetic Speed-beads (Fisher) as described by For analysis of CRISPR/Cas9 induced mutations, PCR fragments flanking the targeted regions were amplified with Eclosion behavior was performed as previously described with slight modifications : monarch perF, 5’-AGTGAAGCGTCCCTCAAAACA-3’; perR, 5’-TGGCGACGAGCATCTGTGT-3’; monarch timF, 5’-GGACGGCAGCGGATACG-3’; timR, 5’-CGCCGTTTCGCACACA-3’; monarch rp49F, 5’-TGC GCAGGCGTTTTAAGG-3’; rp49R, 5’-TTGTTTGATCCGTAACCAATGC-3’; monarch clockF, 5’-CATACCGGTTATAGTCGCACAAGA-3’; clockR, 5’-GATGGGTTCGCTGCAACTG-3’. The near 100% efficiency of each primer set was validated by determining the slope of Ct vs. dilution plot on a 3 × 104 dilution series. Individual reactions were used to quantify each RNA level in a given cDNA sample, and the average Ct from duplicated reactions within the same run was used for quantification. The data for each gene in a given condition (genotype and time of day) were normalized to rp49 as an internal control, and normalized to the mean of one time point within a set for statistics.Brains from adult monarch butterflies entrained to seven LD cycles were dissected under red light on the first day of transfer into DD. Dissections were performed in 0.5 × RNA later (Ambion) to avoid RNA degradation, and brains free of eye photoreceptors were stored at –80° until use. Total RNA was extracted using an RNeasy Mini kit (Qiagen), treated with RQ1 DNase (Promega), and random hexamers (Promega) were used to prime reverse transcription with Superscript II Reverse Transcriptase (Invitrogen), all according to the manufacturers’ instructions. Quantifications of clock gene expression were done using SYBR Green-based real-time quantitative PCR assays with an ABI 7500 Fast Real-Time PCR system (Applied Biosystems). PCR reactions were assembled by combining two master mixes: the first mix contained 5 μl of iTaq Universal SYBR Green Supermix (Bio-Rad) and forward and reverse primers (5 picomole each) per reaction, and the second mix contained approximately 75 ng of cDNA template and the water needed to bring each reaction to a final volume of 10 μl. Each reaction was assembled by consecutively aliquoting each mix into a PCR plate. The monarch P-values were calculated using a Student’s t-test and one-way or two-way ANOVAs using the QI Macros Statistical software for Excel.clk knockout line is available upon request.The monarch cryptochrome 2 (cry2), which encodes the main transcriptional repressor of the monarch circadian clock (clock (clk), which encodes a component of the heterodimeric transcriptional activator complex of the clock  were injected with 0.5 μg/μl of mRNAs encoding the TALEN pair. Given that fertilization occurs at oviposition and the zygote nucleus is formed and starts dividing soon after, injecting shortly after the eggs have been laid likely increases the chances to target the nuclei that will later give rise to germline precursors  survived injection, and all exhibited mosaicism at the targeted site with frequencies of mutated lesions ranging from 3–86%  sex and the male is homogametic (ZZ); 1 progeny. Consistent with clk being linked to the Z chromosome, we found a single mutation per animal tested. All these mutations were microdeletions, and a 4 bp-del mutant that would generate a truncated, nonfunctional protein .To determine the germline transmission rates of TALEN-induced mutations and establish a clk and cry2, for direct comparison.With a more straightforward design and faster construction time, the bacterial-derived CRISPR/Cas9 system has emerged as an alternative to TALENs for the generation of gene knockouts. We tested its efficiency in the monarch butterfly, focusing our study on the genes previously targeted with ZFNs and TALENs, clk, we coinjected monarch eggs with 0.1 μg/μl of an in vitro transcribed single guide RNA (sgRNA) complementary to a 19 bp DNA sequence in the second exon, along with 0.5 μg/μl of mRNA encoding Cas9 . Although these data suggested that the DNA cleavage mediated by sgRNA2 was likely inefficient, they also suggested that when two DSBs were introduced, the occurrence of large deletions was likely favored. Testing whether these large deletions were transmitted to the germline was hindered by the fact the butterflies did not survive to reproductive maturity. However, we were able to provide evidence of germline transmitted indels generated with sgRNA1 in the two larvae that eclosed from two backcrossed founders, presenting the highest rates of somatic targeting .At the site targeted with sgRNA2, we only detected mutagenic lesions in two of the 13 larvae genotyped. However, these legions were large deletions of ∼350 bp , a robust and easily tractable behavior under the control of the brain circadian clock  generated through three independent and consecutive backcrosses, thereby reducing the chances that the phenotype observed result from potential off-target effects. We found that the circadian timing of adult eclosion was disrupted in clk knockouts, while sibling wild-type and heterozygote butterflies eclosed rhythmically, exhibiting a peak of eclosion in the early subjective day at the populational level .The occurrence of a mammalian-like core component in the monarch (CRY2), and the fact that mouse CLK is not required for central circadian clock function . These behavioral and molecular data confirm that monarch CLK is functioning as an essential component of the CLK:CYC transcriptional activator complex of the monarch circadian clock, similar to its Drosophila homolog (To determine how the molecular clock was affected by CLK deficiency, we quantified by quantitative real-time PCR the expression of two core clock components, clk and defined its critical function as encoding a transcriptional activator of the circadian clock used by monarchs during their migration, providing a valuable resource to further our understanding of the clock control of this spectacular behavior.The ability to harness efficient reverse-genetic approaches in butterflies is critical for these species to provide powerful model systems. Even though the use of artificial nucleases has proven to be successful for targeted mutagenesis in two butterfly species, the monarch butterfly and the swallowtail butterfly (cry2 targeting using CRISPR/Cas9 and clk targeting using TALENs. Whether these differences are due to different sgRNAs achieving different mutation rates, or to differences in local DNA accessibility, remains unclear.As previously reported, the use of TALENs and/or CRISPR/Cas9 for nonmodel insect genome editing confers several advantages over ZFNs: target selection is more flexible, their assembly is easier (in vitro cleavage assays (In spite of the broad interest in the use of CRISPR/Cas9 for targeted mutagenesis in nonmodel insects, the general lack of simple and inexpensive assays to quantitatively assess rates of targeting in individual mutants with no visible phenotype has limited the generation of knockout lines to only a few species (In conclusion, the methods we established for the rapid generation of heritable targeted mutations in the monarch butterfly should be widely applicable to other lepidopterans. Nuclease technology will also ultimately enable the systematic dissection of the monarch molecular clock mechanisms and the genetic basis of its long-distance migration. The threat to the North American monarch migration posed by ongoing climate change and habitat loss both across the major flyway in the US and at the overwintering sites in Mexico has prompted national conservation efforts to protect and restore their habitat. Understanding the genetic system underlying the monarch migratory program may help strengthen current conservation strategies.


The induction of oxidative stress by Hg can affect antioxidant enzymes. However, epidemiological studies have failed to establish clear association between dental fillings presence and health problems.To determine whether heavy metals (in hair), antioxidant enzymes (SOD-1) and glutathione levels could be affected by the chronic presence of heavy metals in women who had dental amalgam fillings.55 hair samples  were obtained. All subjects (mean age 44 years) who had dental amalgam filling for more than 10 years (average 15 years). Certain metals were quantified by ICP-MS (Mass Spectrophotometry) in hair  and SOD-1 and Glutathione (reduced form) levels in plasma. Data were compared with controls without amalgams, and analyzed to identify any significant relation between metals and the total number of amalgam fillings, comparing those with four or less (n = 27) with those with more than four (n = 15). As no significant differences were detected, the two groups were pooled .Hg, Ag, Al and Ba were higher in the amalgam group but without significant differences for most of the heavy metals analyzed. Increased SOD-1 activity and glutathione levels (reduced form) were observed in the amalgam group. Aluminum (Al) correlated with glutathione levels while Hg levels correlated with SOD-1. The observed Al/glutathione and Hg/SOD-1 correlation could be adaptive responses against the chronic presence of mercury.Hg, Ag, Al and Ba levels increased in women who had dental amalgam fillings for long periods. Al correlated with glutathione, and Hg with SOD-1. SOD-1 may be a possible biomarker for assessing chronic Hg toxicity. Mercury toxicity varies between different types of mercury and its organic forms are much more toxic than inorganic forms ,3. Although dental amalgam fillings seem to be safe, they are nevertheless a significant chronic contributor to mercury body burden ,5,6. Heavy metals and oligoelements can be detected in human urine, plasma or hair samples by inductively coupled plasma-mass spectrometry (ICP-MS) . However, many epidemiological studies have failed to establish clear association between amalgam fillings presence and health problem . The induction of oxidative stress by Hg is caused in part by the interaction between Hg and antioxidant enzymes . One way that cells remove ROS is by producing proteins such as glutathione and metallothionein that bind to ROS and form more hydrophilic compounds that are easily excreted through Glutathione S-transferases . Copper and zinc-containing superoxide dismutase, (Cu/Zn- cytoplasmic enzyme (SOD-1) metabolizes superoxide radicals to molecular oxygen and hydrogen peroxide, providing defense against oxygen toxicity ,10,11. For this reason, there is a need to establish whether the Hg from dental amalgam fillings can increase heavy metal release and affect glutathione or SOD-1 activity as compensatory mechanism in patients (women).Richardson The present study set out to quantify a plethora of heavy metals levels  in the hair of woman who had dental amalgam fillings for at least 10 years (average 15 years), comparing the data obtained with women control subjects without amalgams.Data were analyzed to find out if the presence of these heavy metal levels was dependent of the total number of dental fillings. For this purpose, subjects (women) were divided into two groups: those with four or fewer dental amalgam fillings  and those with more than four in their mouth ; pooled:dental Amalgam filling group, n = 42 .Reduced glutathione levels and SOD-activity were also analyzed as possible compensatory mechanisms in the body of these woman. Lastly, data were analyzed to discover whether glutathione/SOD-1 activity (plasma) and Zn correlated with levels of heavy metals in subjects’ hair.The present study has been approved by the Institutional Review Board from Murcia University  and following the Declaration of Helsinki. All Subjects in the present study have been properly instructed and they consent to participate by signing the appropriate informed consent paperwork. In addition, all efforts have been made to protect patient privacy and anonymity. CIROM Center has been approved and certificated by AENOR Spain . This study was developed following STROBE guidelines. The women subjects included in this experimental study fulfilled the following criteria:Inclusion criteria Women who had four or less dental amalgam fillings (<4) in their mouth (n = 27); women subjects with more than four dental fillings (>4) (n = 15). All had dental amalgam fillings for at least ten years (average 15 years). Control subjects (women) without dental amalgam fillings were also included in the study (n = 13). All the women were matched in a similar age range . Other criteria were the absence of psychiatric disease, no previous history of DMSA use, subjects untreated (women) with prescription chelators, or any current treatment for iron deficiency (anemia). The women had no history of liver or kidney disease.Physically handicapped women with metabolic diseases or progressive neurological disorders  were excluded, as were those taking regular medication such as stimulants, anticonvulsants, and atypical antipsychotic drugs.The total sample was made up of 42 women, 42 who had dental amalgam fillings for at least ten years (average 15 years) and 13 control subjects (women); all were age-matched (mean age = 44 years). The dental amalgam filling group was divided into two groups: subjects (women) with a total number of amalgams greater than four (>4) (n = 15) and subjects with four or fewer dental amalgam fillings (<4) (n = 27). As no significant relation was found between heavy metal presence and the total number of amalgams in their mouth, the two groups were pooled into a single group . Mercury (Hg) is an essential constituent of dental amalgam fillings (around 50% in weight), together with Ag , Sn (31%) and Cu (28%), and Zn on some occasions. Hair samples close to the scalp were taken from all subjects  and tested to determine these heavy metal levels by inductively coupled plasma-mass spectrometry (ICP-MS) ; Doctor’s Data is a pioneer laboratory in toxicological analysis of heavy metals with over 35 years experience and provides testing to healthcare practitioners. ICP-MS values for heavy metals were reported in μg/g of hair. These patients (women) had a medium/higher sociocultural status.. The assay system consisted of 0.1 mM EDTA, 50 mM sodium carbonate and 96 mM of nitro blue tetrazolium (NBT). In the plate, 470 μl of above mixture was added to 30 μl of plasma and the auto-oxidation of hydroxylamine was observed by adding 0.05 ml of hydroxylamine hydrochloride (pH 6.0). Finally, SOD-1 activity was measured by the change in optical density at 560 nm for 2 min at 30/60 s intervals and normalized as Optical Density (D.O) by protein.SOD-1 activity was measured by means of a modified protocol as published Tiwari V, Chopra K  plus EDTA (5 mM) (pH 8.0) and H3PO4 (25%) were added to the samples. The plasma was centrifuged for 30 min. at 13.000 r.p.m and supernatants were removed. Reduced glutathione levels were determined as follows: 200 μl of 40 mM N-ethylmaleimide and 100 μl of ortho-phthalaldialdehyde were added and fluorescence was measured at 350 m excitation and 420 nm emission, after 15 min of incubation at room temperature (RT).The concentration of reduced glutathione was determined following the procedure described by Hissin and Hilf (1976) Data were analyzed using SPSS software (V17.0) and G Power. Mean and standard deviation were estimated for age and heavy metal levels in study group subjects’ hair and in control subjects (women). The Bonferroni and t Student test (2 tails) were applied to determine possible significant differences between women who had dental amalgam fillings (patients with four or less than four fillings and women with more than four fillings in their mouths) vs controls. Non parametric tests were applied when there was no homogeneity of variance . Differences were considered statistically significant when p<0.05 and considered highly significant when p<0.01.There were changes in heavy metal levels in hair from women with amalgam fillings for more than ten years: Hg, Al, Ag and Ba were higher in the hair of these subjects than in controls . However, both groups (<4 and >4) are not statistically different between them but they are significantly higher than controls .Women with fillings  showed higher Al levels than controls (p<0.029). Women with more than four (>4) fillings had increased Al levels as compared with control subjects (p<0.05) as did women with four or less (<4) (p<0.05). Women with dental amalgam fillings showed higher silver levels than controls (p<0.05) Women with less than four dental amalgam fillings (<4) and the total amalgam filling group showed higher Ba levels than the control subjects p<0.05; .The other heavy metals analyzed  were unaffected by the presence of dental amalgams in all cases . There were no differences for Sb, Be, Bi, Tl, Ti, U or either (data not shown) There were changes in SOD-1 activity, glutathione (reduced form) and Zn levels in women with long-term dental amalgam fillings: correlation between Hg/Al levels and antioxidant markers (SOD-1/Glutathione) SOD-1 activity  and glutZn was found to be significantly higher in women with dental amalgam fillings compared to control subjects without fillings p<0.05; .,15. Ba levels were also seen to increase in women with dental fillings; this feature reflects the findings of another study in which increased Barium (Ba) levels exceeding the local permissible limits were found among dentists . In the present study, Hg and Al levels were correlated between subject with fillings , although Hg, Ag, Al, or Ba levels did not differ significantly between women with or more than four dental fillings in their mouths. These findings suggest that Hg could migrate from the oral cavity . Moreover, other authors have also reported release of mercury from dental amalgam fillings  in agreement with the higher Hg levels observed in our study . A recent study has analyzed levels of heavy metals  in dental pulp and blood samples from subjects with long-term amalgam restorations . The study found heavy metal levels in blood and pulp blood samples in consonance with the higher Hg levels found in our study in hair. Moreover, other authors have also reported the release of mercury from dental amalgam fillings ,18 in agreement with the higher Hg levels (hair) observed in our study. A clinical trial  of 534 children with fillings for more than five years reported higher mercury levels in agreement with the present study in women, . In the study by Saghiri et al. (2014), the long-term presence of dental amalgam fillings (five years) did not induce remarkable changes in mercury levels within dental pulp tissue . However, mercury levels increased in blood circulation five years after the placement of the restoration, which agrees with the highest Hg levels found (hair) in women with amalgam fillings in our study. Any discrepancies in Hg levels between different tissues  can be explained by the different periods the fillings considered in both studies, five years in the Saghiri et al (2014) study compared with at least ten years in the present study . We applied an inclusion criterion of a minimum ten-year presence of dental amalgam fillings in the mouth because chronic exposure to various forms of mercury generated by the amalgam filling is characterized by a very long-lasting latency period of five to seven years before symptoms occur . In addition, women were divided according to the number of fillings: four or less or more than four dental amalgam fillings in their mouths [ Although factors such as the number of years in the mouth, filling size and surface area contribute to the chronic mercury body burden [, heavy metals levels were not different between women with four dental amalgam filling as compared to patients with more than four.Dental amalgams are the main source of the total mercury body burden in humans. This study found that mercury (Hg), silver (Ag), aluminum (Al) and barium (Ba) levels increased in the hair of women who had dental amalgam fillings more than ten years (average 15 years) in comparison with women control subjects without amalgam fillings (p<0.05). The values obtained agree with other studies that have reported 2–12 times higher Hg values in the body tissues of patients with dental amalgam fillings 0). Piggato et al. 2013 reported that monitoring blood and urine is a useful means of identifying individuals with acute exposure to mercury but that the method might underestimate mercury retention and toxicity in tissues and organs [. For this reason, the present study measured Hg levels in hair, since mercury in blood and urine do not fully reflect the actual mercury burden in the human body [. Hg can affect antioxidant enzyme , like SOD-1 and catalase . In fact, Hg produces reactive oxygen species (ROS), especially hydrogen peroxide (H202) and superoxide anion radicals (O2•), which are removed by SOD enzymes  [. As methylmercury (MeHg) affects SOD-1 [, the present study set out to determine whether SOD-1 might be increased among women with amalgam fillings (with a mean age of 44 years). These subjects showed higher increases in glutathione levels than controls, suggesting that Hg can affect oxidative stress by interfering with antioxidant enzyme activities in women [. In our study, a positive correlation between Hg levels and oxidative stress-related SOD-1 activity was also observed , while Al was correlated with levels of glutathione (reduced form) , a finding that agrees with another study in which increased glutathione transferase (GPX3 and GSTM3) expression was observed in the blood of female double-crested Cormorants [. Since increased antioxidant activities have been seen to correlate to high mercury accumulations in the liver and kidneys [, it seems possible that higher glutathione (reduced form) and increased SOD-1 activity might reflect compensatory mechanism to the chronic presence of mercury in women. The increased SOD-1 activity observed agrees with human studies of fish-eating Amazonian communities, which identified an association between fish consumption and other oxidative stress biomarkers  , similarly to the SOD-1 and glutathione levels found in our study. This evidence suggests that glutathione and SOD-1 overactivation are crucial endogenous responses to prevent possible Hg toxicity in people with dental amalgams. Interestingly, Ala16Val MnSOD polymorphism (SOD-2) has been reported in cells exposed to methylmercury in vitro [ In addition, Plecotus auritus (Golden-eared Bat) had altered expression of other antioxidant systems (GSTM3 mRNA levels) associated to high Hg levels (blood) . These increased endogenous antioxidant responses shown by glutathione and SOD-1 upregulation in women are in consonance with studies reporting that SOD-1 acted against methylmercury or cadmium toxicity ¨in vitro¨ [Larus crassirostris (Black-tailed Gull) had high metal concentrations (Mn and Al) in association with changes of antioxidant systems (blood) ,31.Mercury levels in whole blood and urine are believed to be a reliable marker of recent exposure to inorganic and elemental mercury  also rose in people with dental amalgams and SOD-1 is a Cu/Zn dependent enzyme . This could be the reason for which we fail to detect any physical alteration in women with long-term dental amalgam fillings [ Thus, SOD-1 could be a potential biomarker in subjects with dental amalgam fillings. In a retrospective clinical study of medical records (41 patients), Piggato et al. (2013) measured mercury levels in samples of blood (n = 19), urine (n = 19), saliva (n = 20), and scalp hair (n = 17) to investigate the association between mercury levels and cases of multiple chemical sensitivity (MCS) [, MCS  is a chronic condition characterized by an exaggerated body response to toxicants. The values detected for mercury in scalp hair (women) are close to values detected by Pigatto et al 2013  in our study; Hg: 7.6±13.6 μg/L (plasma); Hg 1.9±2.5 μg/L (urine); Hg  = 38.1±52.1 μg/L) [.Al accumulation can induce CNS alteration and lead to dementia although there were no apparent physical alterations in woman with higher Al levels (hair) in the present study . However, some of these revised clinical studies have used insufficient time-frames as well as flawed measures of exposure such as blood or urine levels. In addition, many of them, particularly earlier studies, underestimated the genetic susceptibilities involved in patients [. As far as these authors are aware, the present study has produced the first clinical evidence for higher SOD-1 activity in plasma among women with long-term dental amalgam fillings, who showed higher mercury levels in their hair than control subjects without fillings [. However, data must be interpreted with caution given the limited sample size (n = 55); the authors hope to confirm the results in further clinical trials. Nevertheless, SOD-1 may prove to be a clinically useful marker to determinate heavy metal contamination in humans. In fact, even 30 years after ceasing mercury exposure, dental nurses still show significant adverse health effects [.Finally, recent re-analyses of clinical trials suggest that susceptibility to mercury toxicity differs among individuals and can be influenced by genetic factors underestimated in some clinical trials [Mercury (Hg), Ag, Al and Ba levels were increased in women with dental amalgam for more than years. The observed Al/glutathione and Hg/SOD-1 correlation could be adaptive response against the chronic presence of mercury. The findings suggest that increased SOD-1, and Glutathione levels could be biomarkers for chronic heavy metal toxicity in women with long-term amalgam fillings.


Noroviruses (NoVs) have high levels of genetic sequence diversities, which lead to difficulties in designing robust universal primers to efficiently amplify specific viral genomes for molecular analysis. We here described the practicality of sequence-independent amplification combined with DNA microarray analysis for simultaneous detection and genotyping of human NoVs in fecal specimens. We showed that single primer isothermal linear amplification (Ribo-SPIA) of genogroup I (GI) and genogroup II (GII) NoVs could be run through the same amplification protocol without the need to design and use any virus-specific primers. Related virus could be subtyped by the unique pattern of hybridization with the amplified product to the microarray. By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs. A NoV GII positive specimen was also identified as having co-occurrence of hepatitis A virus. The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene. In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis. Noroviruses (NoVs) are recognized as the leading causative agents of outbreaks and sporadic cases of nonbacterial acute gastroenteritis across all ages in humans, resulting in more than 267,000,000 annual infections worldwide and over 200,000 deaths each year among children under 5 years old in developing countries  of three diverse human enteric viruses including hepatitis A virus (HAV), NoV and coxsackievirus B2 (CXKV B2) from minute amount of starting viral RNAs without using any virus-specific primers. The amplified products were correctly identified by subsequent microarray analysis, displaying high level of reproducibility and fidelity in appropriate sensitivity ranges  and a NoV #186  were used as reference positive materials. Viral RNA was isolated using QIAamp viral RNA mini kit  per manufacturer’s instruction.® pico WTA system  following the manufacturer’s directions. The Ribo-SPIA includes 3 sequential reactions: first a reverse transcription reaction to generate first strand cDNA using a combination of random hexamers and poly-T chimeric primer; secondly a synthesis of DNA/RNA heteroduplex double strand cDNA with DNA polymerase; and thirdly a linear isothermal DNA amplification process in the presence of RNAse H, DNA polymerase, and a SPIA DNA/RNA chimeric primer. The final amplification product is single-strand cDNA (sscDNA) with sequence complementary to the original RNA.Viral RNA amplification was performed using a previously described Ribo-SPIA method  assays were performed on Norwalk and 186, respectively, in a SmartCycler instrument . The GI- and GII-specific primers and probes were used in reactions as previously described .Virus detection and genotyping were assessed using the FDA_EVIR microarray chips described in previous study . The primary microarray data were analyzed with a script based on Affymetrix power tools as described previously  and 186 (GII), respectively, using rRT-PCR. Fold change was measured from ∆Ct value obtained from rRT-PCR results before and after Ribo-SPIA amplification, combined with dilution factor of 10 for each sample tested. As shown in Fig. Evaluation of discriminatory efficiency of the Ribo-SPIA/microarray system was accomplished by using a blinded panel of 22 RNA samples which were isolated from fecal specimens. Two positive reference strains of Norwalk and 186 were also included in the test. The results of microarray analysis are shown in Fig. P = 0.2482). In addition, HAV RNA was also detected by nested RT-PCR in 106 which was NoV GII positive. Sequence comparison between the nested RT-PCR product with other HAV strains revealed that the virus displayed the highest sequence similarity to a subgenotype IB strain HM175/18f , GII.8 (2), GII.1 (2), GII.5 (2) and GII.2 (1) as shown in Fig. Reverse transcription followed by PCR reaction with primer sets designed to amplify specific viral RNA regions is the method of choice to amplify human NoVs prior to downstream molecular analysis. However, high sequence variability of the viral agents posts a challenge to the design of robust universal virus-specific primer sets to amplify various virus variants. Recent studies described the use of multiple GI and GII-specific degenerate primer sets for rRT-PCR to detect a wide range of GI and GII NoVs (Kageyama et al. In comparison to RT-PCR based assays, which require the design and optimization of virus-specific primers for target amplification and only provide presumptive results for the presence or absence of the queried viruses, this sequence-independent amplification method offers several advantages. It obviates the need for an assumption to guild testing suspected viral pathogens with multiple viral-specific primers to amplify targets present in a sample, thus does not require prior sequence knowledge of the viral agent for primer design. Furthermore, a number of viruses cause similar clinical symptoms which make it difficult to choose correct diagnostic analysis under certain clinical hypothesis. In such case, the lack of virus-specific primer sets enables detection of viral agents that might not be found with virus-specific RT-PCR assays. Sample 106 that was presumed negative for HAV infection went merely under NoV RT-PCR test initially. But the microarray analysis here revealed that a subgenotype IA HAV and a GII NoV were simultaneously identified in this sample after Ribo-SPIA amplification Fig. . The preThis study represents the first effort to describe the application of Ribo-SPIA method to amplify the genetically diverse NoVs without using any virus-specific primers for microarray-based detection and genotyping. This sequence-independent amplification linked to DNA microarray analysis allowed identification of multiple NoV genotypes tested in current study. It is expected that this system will also be able to detect other NoV genotypes not examined here since the probe elements derived from those respective viral genomes have already been printed on the microarray chips. Moreover, the use of the sequence-independent amplification protocol eliminates the need for a priori genomic sequence knowledge of the viral agents and thus can extend the range of applications to other RNA viruses.


To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies  and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5)  Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes. Their primary role is to maintain chromosome and genome stability by preventing chromosomal ends from being recognised as double-strand breaks and by protecting them from end-to-end fusion and degradation n hexanucleotide repeats In humans, telomeric DNA is typically 10–15 Kb and is composed of tandem (TTAGGG)In addition, telomeres interact with a number of proteins that can influence chromosome-end integrity and dynamics Dysfunctional telomeres are caused by the loss of telomeric repeats or the loss of protection by telomere-associated proteins and are recognised by many DNA damage response proteins. The recognition of dysfunctional telomeres induces a series of events that compromise the proliferative capacity or viability of the cell, including increased levels of recombination at the chromosome ends, altered gene-expression patterns, chromosome fusions, genome instability, growth arrest and cell death In somatic cells, after many rounds of cells division, telomeres become critically short due to the end replication problem and are recognised as DNA damage Oxidative DNA damage constitutes the majority of DNA damage in human cells One form of DNA damage induced by oxidative stress is the alteration of DNA bases to species such as 8-oxoguanine (8-oxoG), thymine glycol, and 5-hydroxy-methyluracil. When the 8-oxoG lesion is not repaired correctly, it induces single or double strand breaks (SSBs or DSBs) and GC-TA mutation, which may lead to genomic instability During oxidative stress, the possibility of 8-oxoG accumulation within telomeres is enhanced by the high incidence of guanine residues in telomeric DNA sequences Chromosome instability related to telomere dysfunction is mediated mainly by the formation of nuclear anomalies such as micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs), which are biomarkers of genotoxic events and chromosomal instability MN derive from whole chromosomes and chromosome fragments that hang back during anaphase and fail to be included in the daughter nuclei during telophase, while NPBs are nucleoplasmic bridges that originate during anaphase when dicentric chromosomes are pulled to opposite poles of dividing cells Therefore, telomere integrity appears to be a critical element in chromosome stability and telomere shortening, which is induced by an increase in oxidative stress leading to genomic instability and mal-segregation, which both occur very frequently in tumour cells Considering that acute oxidative stress can occur in several states, such as physical activity, inflammation, the tumour microenvironment and cells exposed to radiotherapy, in this work, we aimed to study acute oxidative stress-induced damage specifically in the telomeric region through the analysis of telomeric and genomic base (8-oxoG) modification. Moreover, we sought to understand the role of telomeres in the genomic instability observed after treatment with acute oxidative stress. Furthermore, we studied the relationship between telomere length and genomic instability by analysing telomere length and abnormal nuclear morphologies .2 incubator.Human primary fibroblasts MRC-5, derived from embryonic human lung primary culture , were grown in modified Eagle’s medium (MEM)  supplemented with 10% foetal bovine serum , 10,000 units/ml penicillin and streptomycin 10 mg/ml , 1% L-Glutamine and 1% non-essential amminoacids , at 37°C in 95% air and 5% CO5 cells per culture flask. Subconfluent cells were treated with H2O2 (10vol-3%), in a complete medium, at the final concentration of 100–200 µM for 1 hr at 37°C in 95% air and 5% CO2 incubator. Cells examined after treatment with H2O2 were compared to parallel cultured control cells grown in the medium without H2O2.Cells were sown on plates 48 hrs before treatment at the density of 3×102O2 treatment. After 1 hr treatment, cells were immediately subjected to the assay (time 0) or grown in fresh medium for different recovery times 1, 4, 15 and 24 hrs to evaluate the activation of repair pathways over time and then subjected to the alkaline comet assay. At the end of treatment, before performing comet assay, an aliquot of cells from each sample was dyed with trypan blue in order to evaluate the percentage of cell death (blue stained cells). This percentage never exceeded 20% in all experimental conditions.MRC-5 cells were analysed by alkaline comet asssay as described by Festa et al. In addition we used formamidopyrimidine-DNA-glycosylase (FPG)-modified comet assay to evaluate oxidative DNA damage. FPG is a glycolase that recognizes and specifically cuts the oxidised bases principally 8-oxoG from DNA, producing apurinic sites converted in breaks by the associated AP-endonuclease activity. Therefore, these breaks can be detected by comet assay and give a measure of oxidative DNA damage, enabling us to detect moderate, but still appreciable damage. We followed the procedure described by Collins et al. Slides were analysed using a fluorescence microscope (Leica) equipped with a camera. Seventy comets on each slide, and two slides for each experimental point, coded and blindly scored, were acquired using “I.A.S.” software automatic image analysis system purchased from Delta Sistemi . To quantify the induced DNA damage we used the Tail DNA (TD%), which is a measure of the percentage of migrated DNA in the tail 2O) was also prepared. All samples were set up on ice then incubated at 37°C overnight to allow exhaustive digestion to occur. Five 84-mer oligomers of TTAGGG repeats were designed containing either zero, one, two, four, or eight 8-oxoG bases incorporated into the telomeric tandem repeat sequence. A reverse oligomer was used (an 84-base sequence of 14 CCCTAA repeats) to construct duplex substrates. All oligomers were sourced from GeneWorks .400 ng of gDNA was incubated with 12 units of FPG in 1x NEB Buffer #1. A control incubated with 1X NEB Buffer #1 but without FPG  was then calculated. The number of 8-oxoG in each oligomer was then translated into the number of 8-oxoG bases per kb telomeric DNA through a standard curve established as outlined in O’Callaghan et al. 5 cells per culture flask and left to grow in complete medium for 24, 48, 72 and 96 hrs and collected.After treatment, PBS washed out media and cells were trypsinized and sown at the density of 3×10Chromosome spreads were obtained following 30 min incubation in 30 µM calyculin-A , The Q-FISH tecnique was based on the use of peptide nucleic acid (PNA) telomere oligonucleotides, that generate stronger and more specific hybridization signals than the same DNA oligonucleotides. The resolution of Q-FISH was in the region of about 200 bp t) were determined from semi-log amplification plots (log increase in fluorescence as a function of cycle number) and compared with standard curves generated from serial dilutions of telomerase-positive (tel+) cell extracts (human large cell lung cancer H460). Each sample was analysed in duplicate, and the experiment was representative. Telomerase activity was expressed relative to the telomerase-positive (tel+) sample.The SYBR green RTQ-TRAP assay was conducted as described elsewhere 5 cells per cultured flask. Cells were incubated at 37°C in the presence of 5′-bromo-2′-deoxyuridine  at a final concentration of 2.5×10−5 M 24 hrs before fixation. 5×10−6 µM colchicine was added during the last 5 hrs of incubation. Cells were then collected and metaphase spreads prepared as yet described. CO-FISH, a recombination-based mechanism that analysed the telomere recombination events between sister chromatids, was performed as previously described by Berardinelli et al. 3 probe labelled with Cy3 and then a (CCCTAA)3 probe labelled with FITC . Images were captured with an Axio Imager M1  equipped with a CCD camera . Only T-SCE (Telomere Sister Chromatid Exchanges) events observed with both leading (Cy3) and lagging (FITC) strand probes simultaneously were considered positive. We calculated the T-SCE ratio as the ratio of the frequency of treated samples relative to the frequency in control samples. A total of 1500 chromosomes were scored.After 1 hr treatment, cells were trypsinized and sown at the density of 3×10Cells were harvested by trypsinization and resuspended in 5 ml of complete medium. Total cells were diluted (1∶20) in 0.9% NaCl and enumerated using a haemocytometer (Coulter Counter). Percentage of dead cells was determined by flow cytometer  with propidium iodide permeability exclusion test. Briefly, cells suspension were incubated 5 minutes with 15 µg/ml of propidium iodide and then analyzed on FL3 parameter in log scale. The percentage of propidium-positive cells were determined by an opportune electronic marker. Every data point was assessed in triplicate.After 1 hr treatment cells were plated on petri dish at different density and Bi-nucleated (BN) MCR-5 were obtained adding cytocalasin B  at a final concentration of 3 µg/ml 24 hrs before fixation The analysis of abnormal nuclear morphologies was performed on 1000 BN cells.Experiments were repeated at least three times.A BN cells were scored as previously described by Fenech The statistical tests vary according to the technique used. We performed the Mann-Whitney U test for the statistical analysis of the telomere length (Q-FISH) and the SSBs, measured by the comet assay. We used the binomial probability test for the statistical analysis of the NPBs, MN and NBUDs and CO-FISH analysis. We used the t-test (2 way ANOVA) for the analysis of telomerase activity, for the analysis of telomeric damage and for the comparisons among multiple group in cell growth and viability assay. Significance was accepted for value p<0.05.The standard alkaline comet assay is a sensitive and reliable method used to investigate cellular DNA damage, in particular, SSBs and DSBs.2O2  with telomere-specific primers is an extremely sensitive method to measure the amount of oxidised residues by analysing the abundance of telomeric FPG-sensitive sites within telomeric DNA 2O2 for 1 hr and collected them immediately (time 0), 1, 15 and 24 hrs after treatment. For both 100 and 200 µM H2O2 treatments, we observed a dose-dependent increase of telomeric damage immediately after treatment (p<0.001) that decreased with time up to 24 hrs of recovery  . To achieve this aim, we analysed MN, NBUDs and NPBs eatment) .The results revealed a dose-related increase of ANMs 48 hrs after treatment for both 100  and 200 For biomarkers MN and NBUDs, we observed a fluctuating trend for 100 µM hydrogen peroxide ; conversNPBs have been shown to correlate with telomere defects 2O2  and 1 hr after treatment revealed an increase in damage that was completely rescued 24 hrs later. These results could be interpreted to signify that, as expected, the genomic damage was repaired within this time. In contrast, the analysis of telomeric sequences indicated an increase in DNA damage 1 hr after treatment that decreased over time but persisted at a significant level 24 hrs after treatment for both doses of hydrogen peroxide. The differences between the two techniques can be explained by considering that the comet assay highlights damage in the entire genome and not in the damage site specifically . The persistence of damaged bases in telomeric versus non-telomeric G-rich DNA suggests not only that the telomere structure is susceptible to oxidative stress but also that DNA damage repair at telomeres is less effective than in non-telomeric regions With these assumptions, we focused our attention on telomeres to test whether persistent base damage has an impact on telomere length.Our data revealed no telomere length modulation 24 hrs after treatment, while a significant telomere shortening was observed after 48 hrs. One likely explanation is that the persistent telomeric damage observed 24 hrs after treatment due to the 8-oxoG is not repaired and is responsible for the telomere shortening observed at 48 hrs, indicating that this phenomenon is dependent on replication. At the subsequent times, 72 and 96 hrs, we observed a restoration of telomere length, suggesting that telomere shortening is a transient consequence of acute oxidative stress.Telomere shortening is a well-accepted cause of chromosome instability. For this reason, after demonstrating a change in telomere length, the next step was to investigate the relationship between the telomere shortening observed and chromosome instability through the analysis of biomarkers strictly related to telomere dysfunction such us MN, NBUDs and NPBs The strict correlation between NPBs and telomere length indicates that this biomarker represents a good readout for telomere defects and any consequent chromosome segregation errors. We hypothesised that telomere shortening leads to an increase in the rate of chromosome bridges; when telomere length is recovered, a decrease in the frequency of chromosome bridges is observed .To understand the mechanism leading to restored telomere length, we performed a telomerase activity assay to evaluate the possible involvement of this enzyme in the observed telomere modulation, and our results indicated no contribution. Furthermore, the ALT mechanism, a recombination mechanism between telomere sequences In this work, we demonstrated a link between the oxidation process and abnormal nuclear morphologies. Oxidative stress has been shown to induce 8-oxoG at telomeric sequences. In addition, telomeres are repaired less efficiently than the whole genome It could be interesting to verify these results in other cells, i.e., tumour cells, to test whether telomerase activity can act as a protective mechanism against oxidative stress. Furthermore, to understand if protein alteration also contributes to the telomere shortening observed, the shelterin complex could be analysed.Studies on acute oxidative stress exposition could be very useful considering that certain conditions may be responsible for increased oxidation levels, such as the tumour microenvironment, radiotherapy and some human diseases.