The use of assisted reproductive technologies (ART) has increased steadily. There has been a corresponding increase in the number of ART-related procedures such as hysterosalpingography (HSG), saline infusion sonography (SIS), hysteroscopy, laparoscopy, oocyte retrieval, and embryo transfer (ET). While performing these procedures, the abdomen, upper vagina, and endocervix are breached, leading to the possibility of seeding pelvic structures with microorganisms. Antibiotic prophylaxis is therefore important to prevent or treat any procedure-related infections. After careful review of the published literature, it is evident that routine antibiotic prophylaxis is generally not recommended for the majority of ART-related procedures. For transcervical procedures such as HSG, SIS, hysteroscopy, ET, and chromotubation, patients at risk for pelvic infections should be screened and treated prior to the procedure. Patients with a history of pelvic inflammatory disease (PID) or dilated fallopian tubes are at high risk for postprocedural infections and should be given antibiotic prophylaxis during procedures such as HSG, SIS, or chromotubation. Antibiotic prophylaxis is recommended prior to oocyte retrieval in patients with a history of endometriosis, PID, ruptured appendicitis, or multiple prior pelvic surgeries. The use of assisted reproductive technologies (ART) to overcome infertility has increased steadily in the United States . In 2012Ever since the birth of the first US infant in 1981 using ART, the number of ART clinics and procedures performed has increased [Staphylococcus) when the upper and mid-abdominal skin is incised, or anaerobes and gram-negative aerobes when the skin near the groin or perineum is incised [Although preprocedural or surgical antisepsis has been associated with an overall decrease in infections, contamination of the procedural site is inevitable . Studies incised , 6. The  incised . In gene incised , 9. Tran incised , 8. The  incised .We searched PubMed, EMBASE, and Google Scholar for all English-language publications between January 1980 and July 2015 with the search terms “antibiotics,” “prophylaxis,” “laparoscopy,” “hysteroscopy,” “hysterosalpingography,” “saline infusion sonography,” and “in vitro fertilization.” The authors independently reviewed the preliminary search results and article titles. Of this initial pool, all authors read relevant abstracts regarding antibiotic prophylaxis for gynecologic procedures, specifically in the context of ART. Given the paucity of prospective randomized control trials (RCTs), all study types, that is, case reports, retrospective cohort studies, prospective case-control studies, and other reviews, were included in the analysis.A total of 640 publications were initially identified using the aforementioned search terms. Of these, 24 (3.75%) publications met inclusion criteria. The publications included, by procedure type, were as follows: HSG, 3 (12.5%); hysteroscopy, 7 (29.2%); laparoscopy, 4 (16.7%); SIS, 5 (20.8%); oocyte retrieval, 4 (16.7%); and embryo transfer, 1 (4.2%). The systematic review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines .  FigurePrior to commencing ART, a thorough assessment of the uterus and adnexa is generally performed and often involves bimanual examination and pelvic ultrasonography (US). In addition, previous HSG films should be reviewed for the presence of tubal or uterine abnormalities such as hydrosalpinges and filling defects . To ruleHSG allows imaging of the uterine cavity and fallopian tubes with injection of contrast media using fluoroscopic visualization . The end Chlamydia infection at the time of the procedure [Though uncommon, post-HSG PID complicates roughly 2% of procedures and can have serious implications , 18. A mrocedure . For thiTubal dilation is also recognized as a risk factor for post-HSG PID. A retrospective review of 278 HSG procedures revealed an 11% risk of post-HSG PID in patients with tubal dilation compared to 1.4% in the general population . Further Chlamydia trachomatis and Neisseria gonorrhoeae should be screened and treated prior to the procedure. In patients with no history of PID, HSG can be performed without antibiotic prophylaxis [Based on these findings, patients at risk for lower genital tract infections includingphylaxis . If HSG phylaxis . In patiphylaxis .SIS involves imaging of the endometrial cavity, using real-time US during injection of sterile saline into the uterus . A specuInfection after SIS is a rare but acknowledged complication , 20. The2) are the most commonly used distension media for hysteroscopy. After sterile surgical prepping of the vagina, hysteroscopy is carried out by the grasping of the anterior lip of the cervix with a tenaculum, followed by cervical dilation to the required diameter of the hysteroscope, insertion of hysteroscope, and distention of the uterine cavity with the distension media [Hysteroscopy is the gold standard for evaluating the endometrial cavity in cases of abnormal uterine bleeding and infertility as well as recurrent pregnancy loss , 27. Thion media . Other aon media , 32. Streptococcus D and Staphylococcus aureus (2 cases each). Six patients did not have vaginal cultures performed and no pathogens were isolated in the remaining 6 patients. In the 2 cases of late-onset endometritis, vaginal cultures showed Streptococcus D in one case and polymicrobial flora in the other case. All patients with endometritis were treated with antibiotics.As with any surgical intervention, hysteroscopy carries risk of infection, but, given the abundance of lower genital tract flora, this risk is small, that is, ranging between 0.18 and 0.55% . In one A prospective study has investigated the efficacy of amoxicillin and clavulanate antibiotic prophylaxis in preventing bacteremia during hysteroscopy . In thisn = 523) or placebo (n = 523) preoperatively. The investigators found an overall infection rate of 1.15% (n = 12) with no statistical difference between the study or control groups. Based on these published studies, most professional societies recommend against routine antibiotic prophylaxis for hysteroscopy in the general patient population [In their randomized trial of 631 women undergoing diagnostic hysteroscopy with or without antibiotic prophylaxis, Kasius et al.  found nopulation .2, three 5 mm trocars are placed in the umbilicus, right and left lower quadrants of the abdomen. A 0° or 30° 5 mm laparoscope is placed through the umbilical port. Accessory instruments through the other 5 mm ports are used as needed. Chromotubation of the fallopian tubes at the time of diagnostic laparoscopy may also be carried out [In current clinical practice, a laparoscopy is usually performed for the definitive diagnosis of endometriosis before pursuing ART . In manyOver the past decade, laparoscopy has grown in popularity among gynecologists due to its improved cosmesis and postoperative recovery times when compared to laparotomy. In comparison to conventional laparotomy for benign gynecologic conditions, laparoscopic surgery carries a low postoperative infection rate . LaparosIn 2005, a placebo-controlled, randomized trial of 450 women undergoing laparoscopy was randomly assigned to receive a single dose of a first generation cephalosporin prior to surgery or placebo . No signIn the early days of ART, most oocyte retrievals were performed laparoscopically with general anesthesia . HoweverThe overall incidence of infection after an oocyte retrieval is estimated to be about 0.4% , 49. In The embryo transfer (ET) procedure is perhaps the most critical step of ART . The maiIn addition to technical aspects, ET can be affected by the genital tract microbial milieu . ClinicaIt is important for clinicians to appreciate when antibiotic prophylaxis is indicated and when it is inappropriate. Appropriate antibiotic usage will reduce infectious postoperative complications and minimize the development of antibiotic-resistant organisms. While routine antibiotic prophylaxis is not recommended for the low-risk gynecologic procedures associated with ART, the assessment of individual risk factors remains crucial. For transcervical procedures such as HSG, SIS, hysteroscopy, embryo transfer, and chromotubation, patients at risk for pelvic infections should be screened and treated prior to the procedure. Patients with a history of PID or dilated fallopian tubes are at high risk for postprocedural infections and careful consideration should be given to the risks and benefits of antibiotic prophylaxis in these patients. For transvaginal procedures like oocyte retrieval, antibiotic prophylaxis is recommended in patients with a history of endometriosis, PID, ruptured appendicitis, or multiple prior pelvic surgeries.


Exposure of 4 days post fertilization Ntr larvae to the prodrug metronidazole (Met), which Ntr metabolizes into cytotoxic metabolites, resulted in dose-dependent cell death and axon degeneration. This was limited to the Ntr-expressing sensory neurons, as neighboring glia and motor axons were unaffected. Cell death was rapid, becoming apparent 3–4 hours after Met treatment, and was followed by phagocytosis of soma and axon debris by cells within the nerves and ganglia beginning at 4–5 hours of exposure. Although neutrophils appear to be activated in response to the degenerating neurons, they did not accumulate at the sites of degeneration. In contrast, macrophages were found to be attracted to the sites of the degenerating axons, where they phagocytosed debris. We demonstrated that peripheral glia are critical for both the phagocytosis and inflammatory response to degenerating neurons: mutants that lack all peripheral glia  exhibit a much reduced reaction to axonal degeneration, resulting in a dramatic decrease in the clearance of debris, and impaired macrophage recruitment. Overall, these results show that this zebrafish model of peripheral sensory axon degeneration exhibits many aspects common to peripheral neuropathies and that peripheral glia play an important role in the initial response to this process.Axon degeneration is a feature of many peripheral neuropathies. Understanding the organismal response to this degeneration may aid in identifying new therapeutic targets for treatment. Using a transgenic zebrafish line expressing a bacterial nitroreductase (Ntr)/mCherry fusion protein in the peripheral sensory neurons of the V, VII, IX, and X cranial nerves, we were able to induce and visualize the pathology of axon degeneration  Currently, millions of people suffer from peripheral neuropathies (PN) that arise from numerous causes, including chemotherapeutics, alcohol, toxins, viruses, physical trauma, genetics, and diabetes Axon degeneration is a feature common to many PNs in vivo visualization of cellular processes in real time; transgenic lines allow selective targeting of a specific cell type; and the availability of a drug-based suicide gene technology that permits conditional and selective ablation of targeted cell populations. This suicide system utilizes a combination of the bacterial enzyme nitroreductase (Ntr) and the prodrug metronidazole (Met) that has been used to model other diseases in zebrafish Axon degeneration has been studied in several animal models that use physical trauma  and all efforts were made to minimize the number of animals used and their suffering.Tg(isl:gfp)Tg(p2rx3.2(−4.0):gal4vp16)Tg(UAS:nsfB-mcherry)zdf10foxd3panther (gift from Steve Johnson) Tg(mpx:gfp)  gl22Tg) Tg(p2rx3.2(−4.0):gal4vp16);Tg(UAS:nsfB-mcherry) embryos/larvae, in addition to expression in sensory neurons, there was also variable ectopic expression between individuals within a single clutch. Therefore, larvae were put into separate groups based on Ntr expression, so that individuals with equivalent expression could be compared for a given experiment.All animal husbandry was carried out as described by Westerfield Larvae at 4 dpf were treated with metronidazole  at a final concentration of 1, 5, or 10 mM in fish water. We found 10 mM to give the maximum degree of ablation, so we used this concentration for our studies. Control fish were treated with fish water alone.Larvae were anesthetized with 0.02% tricaine in fish water and transferred to a 96-well plate. Epifluorescent images were taken using a Nikon TE200 inverted microscope equipped with a CoolSNAP HQ digital camera. MetaMorph software  was used to acquire and process images. Cropping and rotating of images was carried out using Adobe Photoshop. For time lapse imaging, larvae were embedded in 0.5% low-melting point agarose containing 0.02% tricaine, and images were taken every 5 minutes.Live embryos/larvae were anesthetized with tricaine (0.01%) and placed in low-melting point agarose (0.5–1.0%) wells present on specially designed tissue culture dishes: these were manufactured by creating a 10 mm hole in the bottom dish and then covering this hole by gluing a glass cover slip on the external surface of the dish. Imaging was performed using a 20x water immersion objective mounted on an Olympus FV-1000 MPE confocal microscope. All images were handled using either Olympus Fluoview or ImageJ software, with final adjustments to contrast and brightness made using Adobe Photoshop.Larvae at 4 or 5 dpf were anesthetized with 0.02% tricaine in fish water and heads were removed with a small scalpel under a dissecting microscope. Microwave assisted larval fixation and embedding were performed as described Larvae at 4 dpf were treated with control PTU water or 10 mM Met for the indicated times, then live larvae were incubated in 10 ug/mL acridine orange in fish water for 20 min in the dark at RT. Larvae were then rinsed three times with fish water, anesthetized with tricaine, and put in a 96-well plate for imaging Larvae at 4 dpf were treated with control PTU water or 10 mM Met for the indicated times, fixed in 4% paraformaldehyde for either 2 hr RT or overnight at 4°C, washed twice for 5 min in PBS, then stained with Sudan Black  for 20 min. Larvae were washed in 70% EtOH until non-specific staining disappears, then rehydrated with a series of EtOH dilutions in PBS plus 0.1% Tween 20 Larvae at 4 dpf were treated with control PTU water or 10 mM Met for the indicated times, stained by incubating in 2.5 ug/mL Neutral Red solution  in PTU water for 6–8 hr in the dark, then rinsed with PTU water and kept in PTU water ON to wash out background staining Tg(mpx:gfp)/Tg(p2rx3.2(−4.0):gal4vp16/UAS:nsfB-mcherry) larvae treated with control PTU water or 10 mM Met were taken every 30 sec for 30 min. Together, these videos spanned a treatment time of 6 hr. The ImageJ plugin MTrackJ Multiple time lapse videos of 4 dpf Sudan Black and Neutral Red stained larvae were embedded on their side in 1% low melt point agarose on an imaging plate and the focus was adjusted to obtain serial images from consecutive planes in the larvae. Using Adobe Photoshop, stained cells were marked in each image and the markings were overlaid to ensure that each cell was only counted once. Marked cells were counted manually in a region that measured a half ear-length from all sides of the ear. Students t test was used to compare means.p2rx3.2:gal4vp16;UAS:nsfB-mcherry) (from here on abbreviated as Ntr) coli nitroreductase (nsfB) protein, fused to mCherry, in the peripheral sensory neurons contributing to the V, VII, IX, and X cranial nerves. It is important to note that due to the variegated expression pattern, only a subpopulation of sensory neurons are targeted. This lack of complete ablation mimics many PNs in which only a subpopulation of sensory axons is affected We have developed a zebrafish model of peripheral sensory axon degeneration, exhibiting characteristics similar to those seen in many sensory PNs, utilizing the nitroreductase/metronidazole (Ntr/Met) suicide system + axons after overnight exposure, demonstrating the high efficiency of this drug in ablating peripheral sensory neurons expressing the Ntr protein )Tg(islet:gfp);Ntr larvae were treated with 10 mM Met overnight, the Ntr-expressing sensory axons appeared punctate, red motile cells were seen in the surrounding tissue, and the ganglia showed signs of degeneration , we crossed Ntr fish to a transgenic line that expresses GFP in branchiomotor neurons ) was used to label dead and dying cells Together, these experiments confirmed that selective ablation of peripheral sensory neurons and their axons could be visualized in real time using the Ntr/Met system in zebrafish.Epifluorescence imaging and AO staining provided strong evidence that axon degeneration and cell death were the result of Met treatment in Ntr larvae. However, these techniques are indirect indicators. Therefore, we wanted to directly verify that these processes did occur by examining tissues at an ultrastructural level using transmission electron microscopy (TEM). 4 dpf Ntr larvae were treated with fish water or 10 mM Met for 4 or 18 hours, then fixed and processed for TEM. Transverse sections of larvae were taken posterior to the ear  and staiTEM images of the transition zone showed a cross section of the vagal nerve bundle in untreated control , 4 h MetTEM images of the vagal ganglia allowed detection of both neuron and satellite glia cell bodies . In contIn addition to the changes in neuronal integrity, the TEM images also revealed phagocytic cells wrapped around presumed dead or dying neurons (electron-dense cell bodies) in the ganglia of Met-treated Ntr fish ) One drawback of SB staining is that it only provides a static snapshot of the number and positions of neutrophils at any given time. To determine if neutrophils were transiently moving through the region of the ganglia and axons, we sought to carry out time-lapse videos of neutrophil movement in response to degenerating axons. To visualize neutrophils, we crossed Ntr fish to a transgenic line that expresses GFP under the control of the promoter of the myeloperoxidase gene gl22Tg ). Overnight treatment with 10 mM Met resulted in the appearance of mCherry-labeled motile cells in both the ganglia and nerve, and confocal imaging revealed that these were co-labeled with GFP  protein panther;Ntr larvae were treated with Met, time lapse imaging showed fluorescent puncta beginning to form at 2–3 hours along the axons and in the plexus . Together, the mpeg:gfp imaging, the NR staining and panther; Ntr data indicated that macrophage recruitment is one response to axon injury in this model.To verify that macrophages were recruited in response to axon degeneration, we examined Met-induced degeneration in a mutant with abnormal macrophage behavior, e plexus , similare plexus ’. Howevepanther;Ntr), Met-treatment still resulted in the appearance of puncta and the degenerating axons/neurons gradually lost their fluorescence  for our experiments. A caveat in the use of this mutant line is that the description of the foxD3 phenotype is based on the absence of transcription factor mRNAs in peripheral glia −/−foxD3; Ntr , indicating that the absence of peripheral glia was not interfering with Met-induced neuronal cell death. Indeed, it appeared that cell death in the mutants occurred to a greater degree, as there were a higher number of dead and dying cells in Met-treated mutants than in Met-treated wild type clutch mates.To determine if this delayed appearance of red motile cells and lack of puncta was perhaps due to postponed or decreased neuronal cell death in the mutants, AO staining was performed on 4 dpf  ganglia . Similar−/−;foxD3 Ntr larvae were treated with 10 mM Met for 4 hours or left untreated, then fixed and processed for TEM. In a nerve cross section of a 4 hour Met-treated −/−;foxD3 Ntr larva   . These fFrom our TEM analysis, we found that mutant larvae had damaged somas and axons with poor membrane integrity after a 4 hour Met treatment; however, in time-lapse videos of these mutants, engulfment of cell debris did not appear until 8–10 hours into Met treatment. In Met-treated wild type clutch mates, damage was apparent by 4 hours and this was concurrent with the appearance of phagocytic cells. These findings suggest that satellite and ensheathing glia are necessary for debris phagocytosis.−/−;foxD3 Ntr larvae to examine macrophage recruitment, and again there was no significant difference in NR stained cells after 2.5 hr between untreated  and Met treated mutants , or 18 hr between untreated  or Met treated mutants . These findings were surprising given the fact that time lapse videos of Met-treated −/−;foxD3 Ntr showed that motile red cells were eventually recruited  and 4 hr-Met treated mutants  (data not shown), indicating that the number of neutrophils did not increase. We also carried out NR staining on ecruited . This deecruited . TherefoDegeneration is a hallmark feature of most PNs. These disorders have a variety of causes and symptoms, affect millions of people, and have no cures. Insults leading to a PN can arise in any type of nerve, can include both myelinated and unmyelinated axons, and often result in axon degeneration and death of the neuron To investigate unmyelinated axon degeneration, we generated a novel zebrafish model. The advantages of this model are that degeneration is targeted specifically to the peripheral sensory neurons, it is conditional, it can be visualized in vivo, the affected nerves are unmyelinated, and many aspects of the organismal response to this degeneration mimic what is seen in the clinical setting. We also demonstrate that the initial response to neuron degeneration is dependent upon the peripheral non-myelinating glia that ensheath both the axons and the cell bodies.While the initial insults that lead to PN in humans are varied, common outcomes include cell death, DNA damage, disruption of microtubules, defects in proteins required for fission and fusion of mitochondria, high levels of reactive oxygen species (ROS) causing oxidative stress, and induction of an inflammatory response Neuronal degeneration and death via apoptosis in human PNs is linked to an increase in neuronal ROS concentrations and mitochondrial dysfunction e.g., chemotherapy-induced sensory PNs Vulnerability of select axons is seen in many human PNs, where the sensory neurons are affected while the motor neurons intact remain intact; Met treatment resulted in neuronal death slightly before, or contemporaneously with, axonal degeneration. Axon damage first appeared as a loss of definition of individual axons within a nerve, followed by the appearance of puncta (phagocytosed debris) and recruitment of immune cells. This is similar to what has been reported for degeneration in other animal models of PN. However, we did not observe a significant increase in the number of neutrophils responding to axon degeneration, which is in contrast to studies in rats and mice that have shown significant neutrophil recruitment in dorsal root ganglia after sciatic chronic constriction injury Neutrophils are not the only immune cell that can be recruited to damaged or dying axon/neurons. Our data showed that macrophages were recruited to degenerating axons, which agrees with the well-established characteristics of Wallerian degeneration and the recruitment of macrophages in models using nerve transection, ligation, and crush + mutants lacking peripheral glia (foxd3−/−) were exposed to Met, no discernible puncta were observed along the axons during treatment, and the appearance of immune cells was dramatically delayed. This was not due to decreased degeneration of sensory neurons/axons, and in fact the opposite occurred: axon degeneration advanced more rapidly in the −/−;foxD3Ntr than in wild type. TEM analysis revealed that axons in mutants treated with Met for only 4 hours contained the large vacuoles and broken membranes that more closely resembled axons from wild types treated overnight with Met. Also, dark puncta located in the axons of Met-treated wild type larvae were not seen in mutants, suggesting that these inclusions were formed due to the presence of peripheral glia. In the ganglia of mutants exposed to Met for 4 hours, degenerating cell bodies had more apparent membrane damage and nuclear defects than equivalently-treated wild type larvae.When Ntr−/−foxd3 larvae, recruitment of immune cells still occurred, although considerably delayed. In wild type larvae, immune cells were recruited starting at 4–5 hours of Met treatment. In the mutants, this initial response has been delayed to more than 8–10 hours of Met exposure. In addition, the response in the mutants is less robust than that seen in their wild-type clutch mates. Since the red motile cells seen in both −/−foxd3 mutants and wild types had similar sizes and movements, such as the speed they migrated and the extension of philopodia, it is assumed that they are macrophages and/or microglia. In addition, no phagocytic cells were detected in TEM images in the ganglia of Met-treated mutants, as was seen in Met-treated wild types. This suggests that these non-myelinating peripheral glial cells have a pivotal role in immune cell recruitment.While puncta were not observed in the axons of treated −/−sox10 zebrafish mutants that lack those glia These findings are supported by previous studies demonstrating that non-myelinating glia express cytokines and chemokines that are known to recruit immune cells in a capsaicin-induced degeneration model One of the major differences between our study and those carried out in mammals is that the nerves we targeted contain only unmyelinated axons. Studies have shown that myelin plays a significant role in immune cell recruitment and in hindering regeneration of axons. Degrading myelin expresses complement C3, which is important in opsonization and phagocytosis of myelin by macrophages −/−;foxD3 Ntr mutant that lacks peripheral glia, we highlight the importance of non-myelinating satellite and ensheathing glia in initiating the inflammatory response and in phagocytosing cell debris. Without satellite and ensheathing glia, neutrophil and macrophage recruitment is postponed, causing a delay in the clearance of harmful debris. This debris can potentially prevent successful regeneration and functional recovery of affected neurons.In this paper, we describe a new model to investigate drug-induced peripheral sensory axon degeneration and hypothesized that non-myelinating glia have a crucial role in the inflammatory response to axon degeneration and phagocytosis of debris. We approached this using the Ntr/Met system to ablate peripheral sensory neurons and assess the inflammatory response, cell death, and the ultrastructure of degenerating axons and cell bodies in both wild type and mutant larvae lacking peripheral glia. Using this method, we found that administering Met caused axon degeneration and neuronal cell death in Ntr-expressing cells, and also elicited an inflammatory response. Using the Some of our findings involving the inflammatory response differed from other studies, such as the lack of significant neutrophil contribution Figure S1Complete ablation of Ntr-expressing peripheral sensory neurons after Met treatment. 4 dpf Ntr larvae treated with A) PTU water (control) or B) 10 mM Met for 18 hours. Confocal microscopy at 5 dpf revealed that vagal nerve axons  remain intact in control animals, but lose all structural integrity when treated overnight with Met (B). Anterior to left, dorsal at top.(TIF)Click here for additional data file.Figure S2Met treatment increases mitochondrial damage in axons. TEM images show examples of healthy (A) and unhealthy (B) mitochondria (arrowheads) present in EM sections of control or treated vagal nerve bundles. Mitochondria were scored and the percentage of mitochondria found as healthy or unhealthy is shown in panel C .(TIF)Click here for additional data file.Figure S3Neutrophil response to degenerating neurons in 4dpf Ntr larvae. A) Sudan Black staining of Ntr treated with fish water (control) or 10 mM Met for 4 hr, (neutrophils labeled with arrowheads). B) SB-stained cell counts in larvae treated with control or 10 mM Met for various times. Average number of SB-stained cells/fish per treatment group shown for incubation times of 3 h , 4 h , 5 h , 6 h , and 18 h . None were statistically significant. C) Neutrophil movement in Mpx:gfp;Ntr larvae treated with fish water or 10 mM Met. Graph shows average velocity (pixels/sec) of neutrophils over time: 0–0.5 h , 0.5–1 h , 1–1.5 h , 1.5–2 h , 2–2.5 h , 2.5–3 h , 3–3.5 h , 3.5–4 h , 4–4.5 h , 4.5–5 h , 5–5.5 h , and 5.5–6 h . Anterior is to the left; dorsal is at the top in all panels. Eye and otic vesicle (OV) are outlined.(TIF)Click here for additional data file.Figure S4TEM images of posterior lateral line nerve in wild type and −/−;foxD3Ntr larvae. A) Cross section of the posterior lateral line nerve (pLL) in 4 dpf wild type Ntr  shows axons surrounded by myelin (arrowheads). B) pLL in 4 dpf −/−;foxD3Ntr  shows axons with adjacent axonal membranes (arrowheads). No myelin is seen.(TIF)Click here for additional data file.Figure S5Loss of foxD3 does not result in an impaired immune response. Tail transections were performed on 4 dpf −/−;mpxfoxD3:gfp or WT clutch mates. Larvae were anesthetized with tricaine and tails were transected with a scalpel at the junction of the body and tail fin. 2 hr after the tail transection, larvae were either imaged for GFP+ cells  or stained with NR to visualize macrophages. A) Uncut tail of WT clutch mate shows the location of the tail transection . A’) Transection site of WT clutch mate showed an increase in neutrophils at the location of the cut. B) Uncut tail of −/−;mpxfoxD3:gfp shows location of tail transection. B’) Transected tail of −/−;mpxfoxD3:gfp also showed an increase in neutrophils at the location of the cut (arrow), similar to that seen in WT clutch mates. C) Neutral Red staining in uncut tail of WT clutch mate. C’) NR staining at transection of WT clutch mate (arrow) showed an increase in macrophages at the location of the cut. D) NR staining at the transection site in a −/−;mpxfoxD3:gfp larva. D’) NR staining of transection site in a −/−;mpxfoxD3:gfp showed an increase in macrophages at the location of the transection (arrow), similar to that seen in WT clutch mates.(TIF)Click here for additional data file.Movie S1Timelapse video of Met-treated Ntr larva. 4 dpf Ntr larva treated with 10 mM Met for 18 hours. Images taken every 12 min. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S2Timelapse video of untreated mpx:gfp;Ntr larva. 4 dpf mpx:gfp;Ntr treated with fish water. Images taken every 30 sec. Video spans a time frame of 30 min taken 4 hours after addition of fish water. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S3Timelapse video of Met-treated mpx:gfp;Ntr larva. 4 dpf mpx;gfp;Ntr treated with 10 mM Met. Images taken every 30 sec. Video spans a time frame of 30 min taken 1.5 hours after the start of Met treatment. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S4Timelapse video of Met-treated panther;Ntr larva. 4 dpf panther;Ntr larva treated with 10 mM Met for 18 hours. Images taken every 5 min. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S5Timelapse video of Met-treated −/−;foxD3Ntr larva. 4 dpf −/−;foxD3Ntr larva treated with 10 mM Met for 23 hours. Images taken every 5 min. Anterior to left, dorsal at top.(MP4)Click here for additional data file.


From previous reviews, there still have been controversies over the effect of metformin (MET) on reproductive function in PCOS patients. The reasons for the inconsistent findings especially lie in the transparency and accuracy of randomized controlled trials (RCTs) reports. However, we could find no data about the quality of RCTs reporting in MET for PCOS. Thus, a retrospective survey related to the quality of reporting in MET for PCOS was conducted.A retrospective survey was conducted by two investigators. Two investigators assessed the quality of overall reporting and key methodological factors reporting using items from the CONSORT 2010 statement.r = 0.589, P < 0.001) between the score of overall reporting quality and year of publication.A total of 39 RCTs were included in full text. The median overall quality score was 9, with a minimum of 2 and a maximum of 13. Good or general reporting existed in 11 items with positive rate of more than or equal to 50%. The median score of key methodological items was 4 with a minimum of 0 and a maximum of 5. Randomization, allocation concealment, blinding, baseline characteristics and intention-to-treat (ITT) analysis were reported in 26 (67%), 19 (49%), 20 (51%), 38 (97%) and 17 (44%) of the 39 RCTs, respectively. After adjustment, the mean overall score increased by about 1.71 for manuscripts with funding source , while it increased by about 3.51 for manuscripts published in one year increment . There was a relatively close, significant correlation (Although the overall reporting quality of RCTs in MET for PCOS has improved over time, reporting of key methodological items remains poor. Reporting of RCTs on MET for PCOS should keep up with the standards of the CONSORT statement. Polycystic ovary syndrome (PCOS) is characterized by chronic anovulation (failure or absence of ovulation) and hyperandrogenism  with clinical manifestations of irregular menstrual cycles, infertility, hirsutism, and acne , which iAdministration of clomiphene citrate (CC) is the standard treatment for PCOS patients with anovulatory infertility. However, clomiphene resistance (failure to ovulate after taking clomiphene) is common, occurring in approximately 15 to 40% of women with PCOS . In 2008Several systematic reviews and meta-analyses -13 have Randomized controlled trials (RCTs) are widely accepted as the ‘gold standard’ for accumulating strong evidence for any health care intervention. Moreover, quality of reporting is essential for guiding journal peer-review decisions and experts’ recommendations, conducting unbiased meta-analysis and influencing our interpretation of evidence . The reaHowever, we could find no data about the quality of RCTs reporting in MET for PCOS. Thus, a retrospective survey related to the quality of reporting in MET for PCOS was conducted. The aim of this study was to assess the overall quality of published articles of randomized trials in MET for PCOS with a special focus on the key methodological items that safeguard against biases, namely appropriate randomization, allocation concealment, blinding, baseline characteristics and analysis according to intention-to-treat (ITT) principle. Secondarily, we also aimed at determining factors associated with better reporting quality.A systematic and comprehensive literature search was conducted with the aim of identifying published prospective RCTs of MET for PCOS. No resources were available to search literature published in languages other than English.The following databases were searched from their inception through February 2013: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL), and CINAHL.Keywords were approved by all the authors and included ‘PCOS’, ‘polycystic ovary syndrome’, ‘metformin’, ‘MET’, ‘clomiphene citrate’, ‘CC’, ‘randomized trials’, ‘RCT’. Eligible articles were identified by successive screening of titles and abstracts. Then the references section of each printed article was screened to identify any additional eligible articles.Types of studies: only RCTs of MET for PCOS were identified and selected for the analysis. Specifically, retrospective, non-randomized, cross-over RCTs, case-control, and quasi-randomized trials, abstracts in conference and case reports/series were excluded.The criteria in terms of PCOS diagnosis had to be consistent with those as follows: oligo- or anovulation, clinical or biochemical signs of hyperandrogenism and polycystic ovaries visible with ultrasound. The RCTs on the effects on MET in patients who received gonadotrophins for IVF and not-IVF cycles were also included. Several different types of interventions were analyzed: MET versus placebo, MET versus CC, MET plus CC versus CC, MET plus CC versus MET. We also included trials in which MET combined with other interventions as the treatment group . The primary comparison was always between MET and the other treatment. Outcomes included live birth rate, rates of ovulation, pregnancy, abortion and discontinuation for adverse events.An overall quality score with 13 items from the CONSORT 2010 statement was used  looked at the title, abstract and methodology of all the published papers to identify them as RCTs. General information  were extracted (by Chun Zhang). Relevant studies were then photocopied with the author’s names, date and institution excluded for following rating by two assessors (Baoying Chen and Jian Liu). Two independent assessors (Baoying Chen and Jian Liu) blinded to each other’s ratings, completed the rating form independently. Cohen’s P ≤ 0.10 in the univariate models were used in a multivariable regression model for selecting significant variables. Variables significant at the 5% level in the final multivariable model were considered as significant predictors. To analyze the relationship between the score of overall reporting quality and year of publications, scatter plot and Pearson correlation analysis were performed.The characteristics of the publications, scores of overall reporting quality and five methodological items were then described by descriptive analysis. To identify factors associated with the overall quality of publications, we used this overall score as the outcome variable and the characteristics of the publications as independent variables which was modeled using linear regression. Only variables that were significant at к-statistics were performed using the SAS software, version 9.1 . Database of RCTs in MET for PCOS are provided in Additional file To identify factors associated with methodological quality, we used this methodological score as the outcome variable in the regression analyses. As the outcome variable can be considered as a count, we relied on a Poisson regression model and adjusted the variance empirically. Descriptive statistical analysis, linear regression analysis, scatter plot and Pearson correlation were performed using SPSS version 20.0 . Analysis of Poisson regression model and Cohen’s The RCTs selection process is outlined in Figure The Journal of Clinical Endocrinology & Metabolism (23.1%), Fertility and Sterility (17.9%) and Human Reproduction (15.4%).The characteristics of RCTs included in the final analysis are described in Table The ratings of overall quality of reporting are listed in Table Inter-rater agreements are reported for each item in Tables Randomization, allocation concealment and implementation, blinding, baseline characteristics and ITT analysis were reported in 26 (67%), 19 (49%), 20 (51%), 38 (97%) and 17 (44%) of the 39 RCTs, respectively (Table P < 0.05), while it increased by about 3.51 for manuscripts published in one year increment   between the score of overall reporting quality and year of publications  statement in 1996 . The CONet al.  developeet al. .Interestingly, we find the overall reporting quality of RCTs in MET for PCOS is improving with the year increasing, which indicates that more and more researchers and editors are realizing the importance of reporting in RCTs due to the widely adoption and promotion of CONSORT. The effect of CONSORT in reporting RCTs of MET for PCOS still needs evaluation. A future evaluation  of the reporting quality after CONSORT endorsement would be useful in assessing the effectiveness of this measure. Meanwhile, our finding that the overall reporting quality based on the CONSORT statement was correlated with funding source, also suggested clinical trials with funding have more capacity to provide assurance for the better quality of study design and reporting of RCTs.There are some limitations to our study. First, we didn’t directly measure RCT methodological quality, because we did not verify the information from the authors or their protocols. As important methodological criteria may be omitted in published reports although adequately carried out, the quality of reporting should be taken only as an imperfect surrogate of true methodological quality. Nevertheless published reports are the major source for clinicians to judge the validity of the results, making the quality of the report essential . Second,Our findings show that the reporting quality of RCTs in MET for PCOS is suboptimal especially in key methodological items. Regarding the crucial methodological issues of blinding, allocation concealment, and analysis by ITT, our results stress the need for researchers involved in RCTs of MET for PCOS to improve the methodological quality of their research through a strengthened international collaboration. Reporting of RCTs on MET for PCOS should meet and keep up with the standards of the CONSORT statement.CC: clomiphene citrate; CENTRAL: Cochrane Central Register of Controlled Trials; CONSORT: Consolidated Standards of Reporting Trials; ITT: intention-to-treat; MET: metformin; PCOS: polycystic ovary syndrome; RCTs: randomized controlled trials.The authors declare that they have no competing interests.BYC: conception and design, data collection and analysis, manuscript writing and final approval of the manuscript. JL: data collection and analysis, critical revision and final approval of the manuscript. CZ: revising the manuscript critically for important intellectual content and final approval of the manuscript. MYL: conception and design, manuscript writing, final approval of manuscript. All authors read and approved the final manuscript.Database of RCTs in MET for PCOS.Click here for file


BLM and CHEK2 are two evolutionarily conserved genes that are somatically altered in a number of tumor types. Both proteins normally function in preserving genome stability through facilitating the accurate repair of DNA double strand breaks. Thus, uncovering synthetic lethal interactors of BLM and CHEK2 will identify novel candidate drug targets and lead chemical compounds. Here we identify an evolutionarily conserved synthetic lethal interaction between SOD1 and both BLM and CHEK2 in two distinct cell models. Using quantitative imaging microscopy, real-time cellular analyses, colony formation and tumor spheroid models we show that SOD1 silencing and inhibition (ATTM and LCS-1 treatments), or the induction of reactive oxygen species (2ME2 treatment) induces selective killing within BLM- and CHEK2-deficient cells relative to controls. We further show that increases in reactive oxygen species follow SOD1 silencing and inhibition that are associated with the persistence of DNA double strand breaks, and increases in apoptosis. Collectively, these data identify SOD1 as a novel candidate drug target in BLM and CHEK2 cancer contexts, and further suggest that 2ME2, ATTM and LCS-1 are lead therapeutic compounds warranting further pre-clinical study.Cancer is a major cause of death throughout the world, and there is a large need for better and more personalized approaches to combat the disease. Over the past decade, synthetic lethal approaches have been developed that are designed to exploit the aberrant molecular origins (i.e. defective genes) that underlie tumorigenesis.  Colorectal cancer (CRC) is a major cause of cancer-related deaths worldwide. In 2015, the American Cancer Society estimates that ~132,700 Americans will be newly diagnosed and ~50,000 additional individuals will succumb to the disease in 2015  and Lung Cancer Screen-1 [LCS-1]) and one chemical mimetic (2-methoxyestradiol [2ME2]) phenocopy the SOD1 silencing results by inducing preferential killing within BLM- and CHEK2-deficient cells. Using semi-quantitative microscopy and RTCA, we show that all three chemicals induce reactive oxygen species (ROS), promote persistent DNA DSBs and potentiate cellular cytotoxicity through an apoptotic mechanism. Finally, we show that drug treatments significantly decrease the number and size of BLM- and CHEK2-deficient cells in 2D colony and 3D tumor spheroid formation assays, respectively. Collectively, our data shows that BLM and CHEK2 are SL with SOD1, and further identify 2ME2, ATTM and LCS-1 as lead candidate compounds warranting further pre-clinical study.In this study, we couple siRNA-based silencing and chemical compounds with semi-quantitative imaging microscopy, real time cellular analyses (RTCA), and biochemical assays to show that BLM-proficient and CHEK2-proficient) cells were purchased from American Type Culture Collection. BLM-deficient and CHEK2-deficient, HCT116 cells were generously provided by Dr. Bert Vogelstein . All HCT116 cells were grown in McCoy's 5A medium (HyClone) containing 10% fetal bovine serum. Immortalized (telomerase) BJ normal human skin fibroblasts, hTERT, were generously provided by C.P. Case [2.HCT116 (.P. Case  (Univers.P. Case . BLM andCells were transiently transfected with siRNA duplexes using RNAiMax (Invitrogen) as described . ON-TARGBLM-deficient, CHEK2-deficient and control (HCT116) cells, and 4,000 hTERT cells were automatically dispensed into each well of a 96-well plate . Cells were transfected in sextuplet (i.e. 6-wells) with either individual or pooled siRNAs targeting BLM, CHEK2, SOD1, and controls (GAPDH and PLK1) as described [t-tests, etc.) were performed. To address reproducibility all experiments were conducted a minimum of 3-times.High-content microscopy was used to evaluate the SL interactions as detailed elsewhere . Brieflyescribed . Cells w50) values were determined. The EC50 values calculated from the BLM- and CHEK2-deficient cells were employed in all subsequent experiments.Standard dose response curves were generated as detailed previously  using a BLM- and CHEK2-deficient cells for 48 h or cells were treated with compounds  for 6 h. Each condition was performed in sextuplet and repeated two additional times. Images were acquired (Cytation 3), signal intensities were determined from raw, unprocessed images (GEN5), and semi-quantitative analyses were performed as described [t-tests, etc.) were performed.ROS were detected using the Image-IT LIVE Green ROS detection kit (Molecular Probes) as detailed elsewhere  with minescribed , 22. AllDirect SL Tests). Each condition was performed in sextuplet and repeated at least two additional times with γ-H2AX and 53BP1 signal intensities determined from raw, unprocessed images. Apoptosis was evaluated by quantifying the total signal intensity of cleaved Caspase 3 as described previously [Semi-quantitative imaging microscopy was employed to evaluate the presence of DNA DSBs as represented by the total signal intensities of two surrogate markers, namely γ-H2AX and 53BP1, as described previously , 22. Brieviously . All datRTCA were performed in quadruplicate and repeated twice using an xCELLigence RTCA-dual plate (DP) system (Acea Biosciences) as detailed . ApproxiBLM-deficient (2 μM) and CHEK2-deficient (5 μM) cells by RTCA. RTCA was performed as above, but with media supplemented with DMSO, 2ME2, ATTM or LCS-1 with or without NAC. All data were imported into Prism where growth curves were plotted.N-acetyl-L-cysteine (NAC) was used to scavenge excessive ROS. The maximum tolerated dose of NAC was determined for BLM- or CHEK2-deficient, or control cells were dispensed into GravityPLUS plates, and tumors were permitted to form for 2-days, whereupon they were released into GravityTRAP plates and cultured in growth medium containing DMSO, 2ME2, ATTM, or LCS-1. Media containing DMSO, 2ME2, ATTM, or LCS-1 were replaced every 2- to 3-days. Cells were incubated for 14-days, at which point nuclei were counterstained with Hoechst 33342, and the medial plane of each tumor spheroid was imaged using a Cytation 3 . ImageJ was employed to determine the maximal diameter of the spheroid [t-tests were performed. Finally, all experiments were performed in sextuplet and repeated at least two additional times.The effect of prolonged DMSO, 2ME2, ATTM and LCS-1 treatments was determined using a standard 28-day colony forming assay as described , and tumspheroid . All datsgs1 and dun1, which are the yeast orthologs of human BLM and CHEK2, respectively. Interestingly, sgs1 and dun1 are SL with several members of the evolutionarily conserved superoxide dismutase pathway, including superoxide dismutase-1 (yeast sod1/human SOD1). This pathway normally functions to remove excess ROS through a two-step process initially regulated by SOD1, which requires Cu2+ as an essential co-factor [BLM and CHEK2-deficient), and would underlie cell cytotoxicity. Accordingly, SOD1 represents a strong candidate to pursue in a cross-species approach designed to identify novel drug targets (i.e. SL interactors) for BLM and CHEK2.To identify novel and lead candidate drug targets to evaluate in a CRC context, we recently employed a cross-species candidate gene approach and identified the SL interactors of yeast genes whose human orthologs are somatically altered in CRC. We queried BioGRID  and ideno-factor , 26. Excsgs1 and dun1 are SL with sod1 [SOD1 is SL with BLM and CHEK2 in humans, we employed an established siRNA-based approach [BLM-deficient (or CHEK2-deficient) and control HCT116 (BLM- and CHEK2-proficient) cells are silenced with either individual or pooled siRNAs targeting SOD1 and controls (GAPDH), and the total number of cells remaining is statistically compared. A SL interaction is expected to result in fewer cells within the deficient cell lines. Before performing the SL tests, we first confirmed BLM and CHEK2 expression levels within all cell lines by Western blots  were performed within the parental, HCT116 cells. In agreement with the above data, Figure BLM or CHEK2 with SOD1 resulted in fewer cells than each condition alone, or the expected number as calculated by a multiplicative model  values calculated for the BLM- and CHEK2-deficient cells relative to controls. In support of chemogenetic SL interactions, the EC50 values for 2ME2, ATTM and LCS-1 are approximately 193-, 54- and 3, 150-fold lower, respectively, in BLM-deficient cells, while they are approximately 1,230-, 1,175- and 1,892-fold lower in CHEK2-deficient cells, respectively. Finally, a fluorogenic ROS indicator coupled with semi-quantitative imaging microscopy and was employed to confirm that 2ME2, ATTM and LCS-1 treatments each induce ROS formation  than that of DMSO treated controls, whereas ATTM treated cells had an increase in cell index (1.4 a.u.) approximately 1-day post-treatment. Approximately 2-days post-treatments, there was a rapid decline in the cell index in each of the condition of treatment that was not readily apparent in the DMSO treated controls. On the other hand, within the CHEK2-deficient cells, 2ME2, ATTM and LCS-1 treated cells showed a markedly high cell index  when compared to DMSO treated controls in 24 hours of treatment with the chemical compounds, which is likely due to a temporary cell cycle arrest and flattening of the cell body (unpublished observation) causing an increase in cellular contacts and thus electrical impedance. Following a temporary arrest as evidenced by a plateau in cell index, there was a rapid and sharp decline in cell index that occurred approximately 2-days (2ME2), 3-days (LCS-1), or 4-days (ATTM) post-treatment. The rapid declines in cell indices for BLM- and CHEK2-deficient cells treated with 2ME2, ATTM and LCS-1 are indicative of strong cytotoxic effects, and are in agreement with the images exhibiting apoptotic hallmarks  were performed. RTCA employs electrical impedance as a measure of cellular proliferation and can easily discern altered growth rates from cell cycle arrests and cellular cytotoxicity. To restrict the therapeutic effect to s Figure  were virs Figure , with eaBLM- and CHEK2-deficient cells treated with 2ME2, ATTM and LCS-1 to those of the vehicle control  was required to induce the cytotoxicity observed above, NAC, an established ROS scavenger was employed in phenotypic rescue experiments. In all instances, NAC addition was sufficient to restore the growth of the l Figure . CollectBLM- and CHEK2-deficient cells treated with 2ME2, ATTM and LCS-1. Since oxidative stress induces various types of cellular damage including DSBs [BLM-  effects 2ME2, ATTM and LCS-1 have on cellular growth both colony formation in soft agar and 3D tumor sphere assays were performed. First, standard colony formation assays were conducted 28-days in which DMSO, 2ME2, ATTM or LCS-1 was supplemented into growth media that was replaced every 2-days. In agreement with the above findings, statistically significant decreases were observed for the total number of s Figure . Next, ts Figure . CollectBLM and CHEK2, which normally encode functions within the HDR pathway. Using two isogenic cell models we demonstrate that SOD1 silencing induces preferential killing within BLM- and CHEK2-deficient cells, and show that these SL interactions are conserved in an unrelated and immortalized cell type. We further show that three chemicals predicted to functionally substitute for SOD1 silencing also induce preferential killing within BLM- and CHEK2-deficient cells in short (5-day), moderate (14-day) and long-term (28-days) assays in both 2D culture and 3D tumor models. Finally, we show that each compound induces ROS, is associated with increases in DNA DSBs, and correlates with increases in apoptosis within BLM- and CHEK2-deficient cells. Accordingly, this study identifies two evolutionarily conserved SL interactions, namely BLM SOD1 and CHEK2 SOD1, and defines SOD1 as a novel candidate drug target in cancers harboring BLM and CHEK2 defects. Finally, our data identify 2ME2, ATTM and LCS-1 as lead candidate compounds warranting further pre-clinical study. Collectively, this study underscores the utility of SL datasets generated in model systems (e.g. budding yeast) to uncover evolutionarily conserved and cancer-relevant interactions that will assist in cancer drug target discovery.In this study, we evaluated the ability of SL interactions initially identified in yeast to predict evolutionarily conserved interactions within a human cell context. Specifically, we sought to determine the ability of SOD1 silencing and inhibition to exploit defects within two genes, 50 values employed in this study are specific to the BLM- and CHEK2-deficient cells, and are significantly lower than those of the controls . With respect to the compounds, ATTM has been employed for unrelated pathologies, while 2ME2 and LCS-1 remain relatively unexplored. For example, ATTM was originally employed to treat copper poisoning in livestock [2+ accumulation. Most recently, ATTM is under investigation for its anti-angiogenic potential based on its ability to prevent endothelial cell homing, cell motility and invasiveness [et al [BLM- or CHEK2-deficiencies). Thus, it is possible that 2ME2, ATTM or LCS-1 treatments in BLM- or CHEK2-deficient/defective tumors may induce anti-angiogenic effects that will synergize with the SL interactions to prevent tumor vascularization while inducing SL killing.SOD1 is highly conserved throughout evolution  and its ivestock , while isiveness –43. In 2s [et al  identifis [et al –47, suggBLM and CHEK2, it is possible that 2ME2, ATTM and LCS-1 may also be effective in familial cancers, in much the same manner that PARP1 inhibitors are being evaluated in the context of familial breast and ovarian cancers harboring inherited defects in BRCA1/2. In a familial cancer context, all individuals are expected to inherit a single wile-type allele and a mutant allele, and it is the subsequent loss of heterozygosity that contributes to the development of familial tumors. Thus, it may be possible to selectively target familial cancers as the non-cancerous  cells are expected to harbor a single wild-type copy of BLM or CHEK2, and produce sufficient protein to render these cells resistant to a SL attack.Although the primary goal of this work is to exploit somatic mutations in et al [i.e. SL interactors) across a set of isogenic cancer cell lines, including the BLM-deficient cells employed in the current study. Interestingly, they did not identify SOD1 as a lead SL interactor, which we attribute to two fundamental differences between our studies. First, Vizeacoumar and colleagues employed a microarray-based approach that is based on the loss of the bar-coded shRNAs within a population of cells, which may not have occurred within the timeframe of their experiment. Second, and perhaps most likely, although SOD1-induced killing is robust within our study, it may not have fallen below the operational threshold required for subsequent validation within their study. Nevertheless, our data complement their findings, and expands the number of validated SL interactors and drug targets of BLM-deficient cells.Over the five past years, there have been numerous successful approaches designed to identify SL interactors of cancer-associated genes, with the ultimate goal of identifying novel drug targets. In 2013, Vizeacoumar et al  performedun1 sod1 SL interaction first identified in yeast [CHEK2. CHEK2 is a particularly attractive and unexplored gene to examine in direct SL tests as it has an established role in HDR [CHEK2 is also of interest as a therapeutic target that can potentiate the cytotoxic effects associated with DNA damage, radiation or chemotherapeutic compounds including camptothecin [TP53-deficient cells to compounds that induce genotoxic stress [et al [TP53-deficient cells were sensitized to doxorubicin, while the TP53-proficient cells were resistant. Although speculative, we predict that the simultaneous depletion of CHEK2 in combination with SOD1 silencing or 2ME2, ATTM or LCS-1 treatments may further exacerbate the sensitivity of TP53-deficient cells to doxorubicin. Thus, targeting SOD1 may have additional therapeutic implications beyond the BLM and CHEK2 genetic contexts explored in the current study.The current study provides the first evidence that the in yeast , is evole in HDR , it is ne in HDR , 51, ande in HDR –55. Howetothecin , PARP intothecin . With thtothecin , anotherc stress , 60. In BLM and CHEK2 contexts examined in the current study, SOD1 may represent a common therapeutic target capable of exploiting many additional genetic defects. Extensive SL data generated in budding yeast [RAD54B, a gene that encodes a helicase functions within the HDR pathway is also SL with SOD1 [RAD54B is somatically altered in ~3.3% of CRCs [BLM and CHEK2 are mutated in up to 4.1% [ATM, BRCA1/2, MRE11, RAD50, NBS1 or RAD51, which are also mutated in CRC and many other tumor types. Furthermore, somatic mutations of SOD1 are rare in cancer, and those few that do occur are mutually exclusive of those occurring for RAD54B, BLM and CHEK2 [RAD54B, BLM or CHEK2, and further support SOD1 as a strong candidate therapeutic target. Finally, SOD1 may also represent an attractive therapeutic target beyond CRC. Although RAD54B, BLM and CHEK2 genes are mutated in CRC, they are also mutated in numerous additional cancers including pancreatic (~16.5% collectively), prostate (~16.7%), melanoma (~15.1%) and endometrial (~8.8%). Thus, studies aimed at exploring SOD1 as a candidate drug target in other HDR-defective gene contexts, and tumor types are highly warranted.A major goal of the current study was to determine whether SOD1 was a shared SL interactor for genes involved in HDR in humans. Beyond the ng yeast , 61, 62 ith SOD1 . While R to 4.1% , respectnd CHEK2 –67. Thes


C-MET proto-oncogene is a tyrosine kinase situated on chromosome 7. C-MET and its ligand hepatocyte growth factor/scatter factor (HGF/SF) play a role in proliferation, differentiation and organ development. C-MET genetic aberrations are found associated with driving tumorigenesis. In this retrospective study, we reviewed molecular analysis data gathered from a cancer institute during a two-year period (2010-2012). Upon detection of tumors harboring c-MET mutations, we determined the status of the other mutations tested and evaluated c-MET expression by fluorescent in-situ hybridization (FISH). Our search resulted in identification of 134 c-MET mutations, 44% of which had mutations of at least one of the other genes tested. No c-MET expression aberrancy was detected in this subset by FISH. Survival amongst the patients with surgically resected metastatic colorectal cancers (CRC) was slightly better in those with only a c-MET mutation compared to those with no mutation detected, although the difference was not statistically significant. When c-MET inhibition becomes an integrated part of chemotherapy practice, our observed frequency of co-mutations will be an argument for utilizing c-MET targeted treatment in combination with other targeted drugs and therapeutic strategies. Larger studies can aid to further parse out c-MET prognostic and therapeutic significance. C-MET tyrosine kinase was first discovered in the1980s [et. al, contains 3 main structural domains: an extracellular Sema domain (Exon 2), a juxtamembrane domain (Exons 14/15) and a tyrosine kinase domain where many of the c-MET mutations have been reported [C-MET, as detailed in a study by Ma ain Exon , a juxtain Exons 4/15 and C-Met signaling has been implicated in a variety of malignancies, including gastrointestinal, genitourinary, breast, gynecologic, pulmonary, head and neck cancers and melanoma. Its genetic aberrations have been reported in a small percentage of this wide variety of tumors ,5. CertaAvailability HGF/SF-MET inhibitors with a range of potencies and specificities has provided a base for assessing their therapeutic value, and initial clinical trials have shown benefit from these treatments in several tumor types. Since c-MET signaling converges with a multitude of pathways, it can lead to activation of several downstream effectors . This diIn this retrospective study we reviewed molecular diagnostic data resulting from a series of mutational analysis during the 2010-2012 period in a cancer center institution. The primary objective was to gain insight into patterns of c-MET mutations and patterns and frequency of co- mutations. We also compared survival outcome in metastatic colorectal adenocarcinoma between those harboring only c-MET mutations as opposed to those with no mutations detected on a 14-gene test.AKT-1, BRAF, GNAQ, GNAS, IDH1, IDH2, KRAS, MET, NRAS, PIK3CA, RET). Mutation detection was carried out utilizing a 9-well based 11-gene mutation hotspot screen developed by our laboratory using Massarray® platform . Briefly, DNA was PCR amplified and subjected to single base-primer extension using iPLEX Gold kit and analyzed on MALDI-TOF mass spectrometry . The regions of interest were PCR amplified using 10ng of DNA per well. Subsequently PCR products were treated with shrimp alkaline phosphatase to dephosphorylate unincorporated nucleotides followed by locus specific single base extension with mass modified di-deoxy nucleotides (ddNTP).We reviewed the molecular analysis report from over 2000 solid tumors undergone mutation testing at MD Anderson during 2010-2012. Cases  were predominantly high stage solid tumors where the mutational analysis was performed to assess for considerations of targeted additional therapy. These tests included a custom designed mutation analysis panel as described previously (Leuk Lymphoma 54(1):138-44; 2013). This quantitative primer-extension MALDI-TOF assay (PE-MALDI) interrogated a total of 88 hotspot mutations in 11-genes  tissue sections using the PicoPure DNA Extraction Kit  following manufactures instructions. In the c-MET mutated subset, patient's demographics, tumor types and stage, SNP patterns, their distribution amongst tumor types, secondary tumors and incidences of co-mutations were recorded . The mass of the products of single-base extension were analyzed by MALDI-TOF for SNP detection. In addition, the exons 18 to 21 of the Additionally, we reviewed the c-MET FISH results when available, to examine whether there is an association of MET amplification with presence of genetic aberration. MET FISH was performed in the clinical cytogenetics diagnostic lab at MD Anderson Cancer center, following the standard clinical procedure established in the laboratory. In brief, glass slides from formalin-fixed paraffin embedded tissue (FFPE) are pre-treated and hybridized with the MET and chromosome 7-centromere FISH probe mixture . FISH results were scored and reviewed on a fluorescence microscope. A total of 60 cells were scored. MET amplification was considered positive if the ratio of average MET to average of CEN 7 was equal or greater than 2.0 or more than 20 copies of MET signals with clusters noted in >10% of the cells.Upon review of the molecular data, we found 134 c-MET mutations, involving 133 tumors (2010-2012), which correspond to 6-7% of the total number of tumors. 5 different c-MET point mutations were detected by DNA sequencing (Table 44% (59 of 134) cases with c-MET mutation had mutations of at least one of the other 13 genes in the tested  with 9 % (12 of 134) having 2 or more co-mutations. These comutated genes included p53, KRAS, NRAS, BRAF, PIK3CA, IDH1 KIT, AKT and EGFR, the distribution of which corresponded to trends generally associated with each tumor type. Save for the one case with Y1253D aberration and co-mutations (1/1=100%), the highest rate of co-mutations was observed with R988C aberration, corresponding to 76% (10/13). The next highest co-mutation rate was with T1010I, corresponding to 37% (14/38) closely followed by N375S at 35% (29/81). No co-mutation was clustered with codon Y1248H. This patient also had history of squamous cell carcinoma and prostatic adenocarcinoma.In cases with metastatic, surgically resected colorectal percentages were detected: c-MET 9.5%, KRAS 43%, PIK3CA 18%, NRAS 4%, and BRAF 8%. The remaining mutations included P53, EGFR, KIT, and IDH mutations.3% of tumors had no mutation detected on analysis. Neither the c-MET mutated nor the subset of cases with no mutation detected showed overexpression of c-MET by FISH, and targeted c-MET treatment was not utilized in their treatment. Those whose sole mutation detected on the panel was a c-MET mutation (n=14) had a better survival compared to subjects with no mutations detected in the analysis (n=24). However, this difference was only marginally significant (p=0.057), which may be due to limited sample size. Results are summarized in tables C-MET receptor tyrosine kinase and its physiological ligands HGF/SF are associated with diverse cellular response upon activation. Ligand mediated dimerization triggers phosphorylation of c-MET and response of cytoplasmic effector proteins including GAB1, GRB2, phospholipase C and SRC. C-MET is intimately associated with pathways including RAS-MAPK, PI3K-AKT and Wnt .The role of c-MET in tumorigenesis through genetic aberration, up regulation of cellular cross talk, and roles in metastasis and activation of cancer stem cells is an area of ongoing research. The evidence for it playing a role in oncogenesis is mounting as not only its somatic mutations have been found in many cancers, germ line c-MET point mutations are present in subset of tumors such as papillary renal cell carcinoma. In parallel a subset of genetic mutations are found as polymorphic variants. Although in our study we did not find upregulation in MET gene expression by FISH analysis, we did detect missense point mutations that through their altered gene product can change the functionality of the c-MET receptor.et. al. to find N375S as a passenger and not a driver mutation [The most commonly identified mutation in our series was the N375S of the extracellular SEMA domain. This aberration results in the medium sized polar amino acid asparagine being replaced by the smaller polar amino acid serine and has been found as a germ line mutation in a cohort of patients with non-small cell lung cancer (NSCLC) . A latermutation . These smutation . In compmutation ,10. It iDespite evidence supporting the T1010I mutation's importance in oncogenesis, not every study confirms this role. This mutation, in the cytoplasmic juxtamembrane domain, results in the substitution of the medium sized hydrophobic amino acid threonine with the medium sized polar amino acid isoleucine. It has been shown to increase growth factor independent proliferation and motility in vitro in tumor cell lines . WaseniuThe other c-MET mutation R988C, affecting the cytoplasmic juxtamembrane domain, results in the substitution of the large polar amino acid arginine with the small polar amino acid cysteine. Studies on this mutation are also conflicting as some demonstrate it resulting in gain a function in SCLC cell lines , while oThe two remaining mutations detected are both affecting the kinase domain. The Y1248H mutation results in the substitution of the large non-polar aromatic amino acid tyrosine with the medium sized polar amino acid histidine. It is a known mutation found in papillary renal cell carcinoma and reported to have a transforming ability ,15. ThisThe discrepancies between studies on whether a particular mutation is transformative may be explained if the same aberration results in different cell phenotypes when affecting different cell lineages. To support this notion, a recent study indicates that overexpression of c-MET associated with a worse prognosis in esophageal adenocarcinoma and not in head and neck cancers . DespitePolymorphisms of c-MET which on their own may lack transformative ability may still play a role in promotion of tumor development and metastasis in combination with other mutations. Its relevance may be closely tied to co- mutation rather than the sole effect of this mutation alone. According to Lee et al., activated c-MET has been shown to be a predictor of poor survival when in combination with other mutations in CRC. The authors identified that clustering of activated c-MET in conjunction with PIK3CA mutation predicts a worse survival outcome . The effAs stated previously, a subset of c-MET aberrations can play a role in response to chemotherapy by rendering the receptors resistant to drug therapies targeting c-MET. This would clearly effect the treatment response by decreasing the response to the drug. In EGFR mutated NSCLC, MET amplification has been demonstrated as an escape pathway and a mechanism of EGFR TKI resistance . Also, iCurrent literature empirically suggests that c-MET aberrations (mutations/protein over expression) are more commonly noted in tumor clones with potential to spread. Activating c-MET aberrations have been found selected in association with nodal metastasis or invasive fronds of the primary tumors. This increased prevalence has been theorized to involve c-MET's signaling function in angiogenesis and cell movement ,20,21. Ahttp://clinicaltrials.gov/ct2/results?term=met). Most of these trials investigate the effects of c-MET inhibitors, usually in combination with other tyrosine kinase inhibitors or other chemotherapy drugs.Diversity of proposed neoplastic mechanisms has led to producing a wealth of inhibitors targeting various aspects of the c-MET pathway. Currently clinical trials are underway involving c-MET and cancers including NSCLC, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, triple negative breast adenocarcinoma, gastric or esophageal adenocarcinoma, and anaplastic large cell lymphoma, to name a few (Our study did not find a survival disadvantage for patients with resected metastatic CRC harboring only c-MET mutation compared to those with none, but rather a marginally significant survival improvement. Although the small study population may limit analysis, we did not find a clear-cut adverse prognostic relevance attributable to c-MET mutation alone, when unaccompanied by a known oncogenic mutation. Further studies may aid in establishing this concept.


BLM, which encodes a DNA helicase that functions in DNA double‐strand‐break repair, make up the vast majority of Bloom syndrome mutations, with only 13 single amino acid changes identified in the syndrome. Sequencing projects have identified nearly one hundred single nucleotide variants in BLM that cause amino acid changes of uncertain significance.Bloom syndrome is an autosomal recessive disorder characterized by extraordinary cancer incidence early in life and an average life expectancy of ~27 years. Premature stop codons in BLM variants incapable of complementing certain defects of Bloom syndrome cells, making them candidates for new Bloom syndrome causing mutations, we characterize a new class of BLM variants that cause some, but not all, cellular defects of Bloom syndrome. We find elevated sister‐chromatid exchanges, a delayed DNA damage response and inefficient DNA repair. Conversely, hydroxyurea sensitivity and quadriradial chromosome accumulation, both characteristic of Bloom syndrome cells, are absent. These intermediate variants affect sites in BLM that function in ATP hydrolysis and in contacting double‐stranded DNA.Here, in addition to identifying five BLM variants that are hypomorphic which, instead of causing Bloom syndrome, may increase a person's risk for cancer or possibly other Bloom‐syndrome‐associated disorders, such as type‐2 diabetes.Allele frequency and cellular defects suggest candidates for new Bloom syndrome causing mutations, and intermediate  BLM) is a rare chromosome breakage disorder characterized by short stature and an extraordinary predisposition to a broad range of cancers, often multiple, early in life  into noncrossover products   in the human BLM gene for those that impair BLM function as estimated by the hypersensitivity of cells to HU (Mirzaei and Schmidt Saccharomyces cerevisiae) and the C‐terminal 800 residues of human BLM to which 41 nonsynonymous SNPs from the Short Genetic Variations database (dbSNP) map. We ranked these 41 coding SNPs and two additional SNPs reported in the literature   suggests that they are insufficient for full‐scale Bloom syndrome, but their lower functional activity may lead to an increased cancer risk later in life or an increased risk for developing other symptoms of Bloom syndrome, such as type 2 diabetes mellitus or fertility problems. Here, we have quantified functional defects of cells expressing these new BLM alleles, with an emphasis on the first three hypomorphic BLM allele candidates  by assessing chromosomal abnormalities, their ability to respond to genotoxic agents and their ability to repair DSBs.There is some evidence that heterozygosity for 2. GM08505 cells were plated 24 h before transfections at approximately 2 × 104 per cm2. BLM cDNA cloned into pcDNA3 vector and mutated at stated sites using site‐directed mutagenesis was transfected using Polyfect . Stable clones were selected in the presence of G418 (750 μg/mL). Clones were maintained in the presence of G418 (350 μg/mL). BLM cDNA in pcDNA3 was a kind gift from Dr. Ian Hickson, University of Copenhagen. Cell lines expressing the BLM variants evaluated in this study are listed in Table GM08505 is an SV40‐transformed skin fibroblast cell line established from a patient with Bloom syndrome , followed by nuclear extraction in 20 mmol/L Tris pH 7.4, 10 mmol/L KCl, 0.4 mol/L NaCl, 1 mmol/L EDTA, 50% glycerol, 0.6 mmol/L BME, 1 mmol/L PMSF, and protease inhibitor cocktail (Pierce). Mouse monoclonal antibody BLM‐F5  was used to detect BLM and a monoclonal tubulin antibody  was used as a loading control.Nuclear extracts were prepared from exponentially growing cells to detect BLM expression. Cells were lysed in 20 mmol/L Tris pH 7.4, 10 mmol/L KCl, 1 μg/mL colcemid for 1 h. Chromosome spreads were prepared after hypotonic treatment with 75 mmol/L KCl and fixation with 3:1 (vol/vol) methanol‐acetic acid. Metaphase spreads were differentially stained using Hoechst 33258  and Giemsa . Images of intact metaphases were acquired on a Zeiss Axiovert 100 deconvolution microscope with a Plan‐Apochromat 100x/1.40 Oil DIC M27 objective Zeiss, Thornwood, NY. For each cell line, a minimum of 1500 chromosomes were analyzed. The mean ± SD is reported.Sister chromatid exchanges (SCEs) were quantified using protocols described previously  or drug‐free medium was added. The medium was removed after 48 h and cells were washed with PBS before colonies were allowed to grow in fresh complete growth medium for up to 4 weeks. Colonies were fixed in 3:1 (vol/vol) methanol‐acetic acid and stained with 0.01% crystal violet. Colonies with more than 50 cells were scored as survivors.μmol/L CPT for 1 h, released into fresh media, and harvested at various time points . To detect γH2AX, chromatin fractions were prepared from nuclear extracts using 0.5 mol/L HCl, 50% glycerol, 100 mmol/L β‐mercaptoethanol and then neutralized using NaOH in 40 mmol/L Tris pH 7.4 with protease inhibitor cocktail (Pierce)  and polyclonal histone H3 antibody (Abcam). Changes in γH2AX levels with respect to histone H3 levels were quantified using ImageJ . Images were acquired on an UltraVIEW VoX Life Cell Imager  equipped with a Zeiss Axiovert 200 and a 20x/0.8 M27 Plan‐Apochromat objective using Volocity software. At least 50 comets were imaged for three independent cell lines for a minimum of 150 comets at each time point. The mean of the tail DNA percentage was calculated using OpenComet , were constructed and analyzed for DNA‐damage sensitivity using a clonogenic  assay  exhibit a spontaneous, approximately 10‐fold increase in the frequency of SCEs compared to cells from unaffected persons; they also present with quadriradial chromosomes in metaphase spreads, are hypersensitive to HU, and show a delay in activating the DNA damage response after exposure to the topoisomerase poison CPT. To determine the ability of eight SNPs that cause single amino acid changes in BLM   than in cells expressing wildtype BLM, indicating reduced BLM activity.The decreased ability of BLM‐deficient cells to dissolve intermediates of HR as noncrossovers is thought to be a cause of the increased frequency of exchange of genetic material between sister chromatids, which can be visualized by differential staining with Hoechst/Giemsa after two rounds of cell division in the presence of the thymidine analog BrdU  Fig. A, but leIn contrast with genetic exchange between sister chromatids, recombination between homologous chromosomes can give rise to LOH; it can thus be considered mutagenic and a driver of tumorigenicity in Bloom syndrome. Such recombination events between nonsister chromatids of homologous chromosomes and, potentially, nonhomologous chromosomes are captured in metaphase spreads as quadriradial chromosomes Fig. C. Quadriμmol/L) induces replication‐specific DSBs , whereas Bloom syndrome cells died. To test if the marked delay in γH2AX accumulation and elimination after CPT removal in cells expressing P868L corresponded to unrepaired DSBs, we performed a neutral comet assay  and then after 1, 24, and 48 h. In wildtype cells, DNA breaks were significantly reduced over the 48 h time course, with 23% and 14% of DNA damage remaining after 24 and 48 h respectively. In blm‐P868L cells twice as much DNA damage  was still unrepaired after 48 h, indicating a lag of DNA repair of at least 24 h. This presents a significant loss in DNA break repair activity of the P868L variant, but not as great as that of Bloom syndrome cells where the amount of unrepaired DNA breaks after 48 h was three times higher  than in wildtype cells.At low concentrations <50 nmol/L) okadaic acid selectively inhibits the 0 nmol/L 2+ and single‐stranded DNA and, eventually, to hydrolyze ATP. The null phenotype exhibited by the five BLM variants can be explained by the location of the mutated residues in or near these conserved helicase motifs. These new null alleles are the first to inactivate BLM by mutation of residues in or near motifs I (P690L), Ia (R717T), VI (G972V), and the conserved aromatic loop , whereas the known Bloom syndrome‐associated amino acid changes map to motifs 0 (Q672R) and IV  or fall outside of the conserved motifs  , that exhibits some, but not all, defects of Bloom syndrome causing mutations and does not, based on its relatively high allele frequency, cause Bloom syndrome. The frequency of P868L in the human population is 5.13%, ranging from 0.6% in the Japanese to 12.3–15.3%  in the population of African descent in the South Western United States (ASW) , which is conserved in members of the RecQ helicase family from bacteria, to yeast, to humans. It is still unclear how the WH domain contributes to BLM function since BLM lacking this domain still possesses helicase activity  mutations. G1120R is the first BLM missense mutation from the human population that maps to the WH domain and impairs BLM function. The WH domain is conserved structurally, but not at the amino acid level. We found that one of the few exceptions is the first glycine (G1120) in the loop between the second and third β‐sheet that precedes helicase motif II  required for Mg2+ binding and ATP hydrolysis , slower accumulation and markedly delayed elimination of γH2AX after genotoxin exposure, and inefficient repair of replication‐dependent DSBs), but not others  , and the lesser, but significant, delay in cells expressing the P868L, G1120R, or R791C variants may be indicative of dysregulation of DSBR, resulting in increased utilization of SSA or HR between homologs, which are prone to deletions and LOH respectively. In contrast with H2AX phosphorylation and its effects on DSBR, the signaling of γH2AX elimination and the consequences of persistence of γH2AX, as seen in cells expressing fully or partially defective BLM variants in this study, are less well‐understood, but involves direct dephosphorylation by PP2A‐B56ε  elimination of cellular γH2AX is associated with delayed and, possibly, error‐prone DNA damage repair as well as delays in restoration of chromatin integrity and the cell cycle.Taken together, we have identified five new BLM missense mutations that exhibit defects of the Bloom syndrome associated s) Table , while ps) Table . Thus, tin silico predictability of the impact of SNPs on BLM function  expressing wildtype BLM and BLM variants to replication‐dependent DNA breaks induced by exposure to 1 μmol/L CPT for 1 h.Figure S2. Effect of PP2A inhibition on γH2AX accumulation in Bloom syndrome cells expressing wildtype BLM and hypomorphic BLM variants.Click here for additional data file.


As for all materials, the performance of concrete is determined by its microstructure. Its microstructure is determined by its composition, its curing conditions, and also by the mixing method and mixer conditions used to process the concrete. This paper gives an overview of the various types of mixing methods and concrete mixers commercially available used by the concrete industry. There are two main types of mixers used: batch mixers and continuous mixers. Batch mixers are the most common. To determine the mixing method best suited for a specific application, factors to be considered include: location of the construction site (distance from the batching plant), the amount of concrete needed, the construction schedule (volume of concrete needed per hour), and the cost. Ultimately, the quality of the concrete produced determines its performance after placement. An important measure of the quality is the homogeneity of the material after mixing. This paper will review mixing methods in regards to the quality of the concrete produced. Some procedures used to determine the effectiveness of the mixing will be examined. As for all materials, the performance of concrete is determined by its microstructure. Its microstructure is determined by its composition, its curing conditions, and also by the mixing method and mixer conditions used to process the concrete. The mixing procedure includes the type of mixer, the order of introduction of the materials into the mixer, and the energy of mixing (duration and power). To control the workability or rheology of the fresh concrete, for example, it is important to control how the concrete is processed during manufacture. In this overview, the different mixers commercially available will be presented together with a review of the mixing methods. Further, the advantages and disadvantages of the different mixers and mixing methods and their application will be examined. A review of mixing methods in regards to the quality of the concrete produced and some procedures used to determine the effectiveness of mixing methods will also be given.To determine the mixing method best suited for a specific application, factors to be considered include location of the construction site (distance from the batching plant), the amount of concrete needed, the construction schedule (volume of concrete needed per hour), and the cost. However, the main consideration is the quality of the concrete produced. This quality is determined by the performance of the concrete and by the homogeneity of the material after mixing and placement. There should be a methodology to determine the quality of the concrete produced, but only few methods and only one attempt of standardization were found in the literature. The methodology to determine the quality of the concrete mixed is often referred to as the measurement of the efficiency of the mixer. The efficiency parameters of a mixer are affected by the order in which the various constituents of the concrete are introduced into the mixer, the type of mixer, and the mixing energy (power and duration) used.There are two main categories of mixer: batch mixers and continuous mixers. The first type of mixer produces concrete one batch at a time, while the second type produces concrete at a constant rate. The first type needs to be emptied completely after each mixing cycle, cleaned (if possible), and reloaded with the materials for the next batch of concrete. In the second type, the constituents are continuously entered at one end as the fresh concrete exits the other end. The various designs of each type of mixer will now be discussed.Two main types of batch mixer can be distinguished by the orientation of the axis of rotation: horizontal or inclined (drum mixers) or vertical (pan mixers). The drum mixers have a drum, with fixed blades, rotating around its axis, while the pan mixers may have either the blades or the pan rotating around the axis.non-tilting drum;reversing drum;tilting drum.All the drum mixers have a container with a cross section similar to that shown in The non-tilting drum mixer implies that the orientation of the drum is fixed. The materials are added at one end and discharged at the other .3 [The reversing drum  is simil3 .The truck mixers belong to the reversing category of drum mixers. The driver of the truck can control the speed of rotation with a clutch in the cabin. The speed depends on whether the concrete has been well mixed prior to being placed in the truck or whether the truck has to do most of the mixing. Typically the speed for mixing is 1.57 rad/s (15 rpm), while the transport of pre-mixed concrete uses only 0.2 rad/s (2 rpm) to 0.6 rad/s (6 rpm) [3) both in the laboratory and in the field [In a tilting drum mixer , the inche field .All pan mixers work on basically the same principle : a cylin3), the blades are lifted and the pan can be removed to empty the mixer.To discharge the mixer, the pan is usually emptied through a trap on the bottom. For small mixers  and/or small deliveries. A major use of these types of mixers is for low slump (non flowable ) concretIn describing the mixing process, the mixer hardware is only one of several components. The mixing process also includes the loading method, the discharge method, the mixing time, and the mixing energy.The loading method includes the order of loading the constituents into the mixer and also the duration of the loading period. The duration of this period depends on how long the constituents are mixed dry before the addition of water and how fast the constituents are loaded. The loading period is extended from the time when the first constituent is introduced in the mixer to when all the constituents are in the mixer. RILEM   divides The loading period is important because some of the concrete properties will depend on the order in which the constituents are introduced in the mixer. It is well known that the delayed addition of high range water reducer admixture (HRWRA) leads to a better dispersion of the cement. The same workability can be thus be achieved with a lower dosage of HRWRA . UnfortuVery often, the mixing time is defined as the time elapsed between the loading of the first constituent to the final discharge of the concrete. RILEM  took anoThe discharge from the mixer should be arranged so that it increases productivity (fast discharge), and it does not modify (slow discharge) the homogeneity of the concrete. For instance, if the discharge involves a sudden change in velocity—as in falling a long distance onto a rigid surface—there could be a separation of the constituents by size or, in other words, segregation .The energy needed to mix a concrete batch is determined by the product of the power consumed during a mixing cycle and the duration of the cycle. It is often considered, inappropriately, a good indicator of the effectiveness of the mixer , 10. TheAs it has been pointed out, the variables affecting the mixing method are numerous, not always controlled, and not a reliable indicator of the quality of the concrete produced. There is, therefore, a need for a methodology to determine the quality of the concrete produced as an intrinsic measure of the efficiency of the mixer. The concept of “mixer efficiency” is used to qualify how well a mixer can produce a uniform concrete from its constituents. RILEM  defines workability of the fresh concrete as defined by the slump;density of the concrete;air content;compressive strength.Since the macroscopic properties of concrete are affected by its composition, it is conceivable that the homogeneity of the concrete produced could be monitored by measuring the performance of specimens prepared with concrete taken from different parts of the mixer or at different times during the discharge. Properties that are often considered areA disadvantage of this method is that it is indirect. It does not directly show that the concrete is homogeneous but only assumes that any potential inhomogeneity affects the properties considered. In addition, it is possible that either the measurement methods selected are not sensitive enough to local changes in composition, perhaps because the samples are too large, or that the properties selected are intrinsically not affected by inhomogeneity. The consistency in the properties is a useful guide but not a definitive indicator of product homogeneity. It can give a false sense of security about the mixing method used.A more direct method to determine the efficiency of a mixer would be to measure the homogeneity of the concrete. This method does not rely on an assumption about the dependency of macroscopic properties on the concrete composition. The measure of the concrete homogeneity can be achieved by determining the distribution of the various solid constituents such as coarse and fine aggregates, mineral admixtures, and cement paste throughout the mixture. However, there are no standard tests to determine homogeneity. Nevertheless, the analysis of samples of concrete taken in various parts of a mixer or at various times during the discharge is usually accomplished by washing out the cement paste and then by sieving the aggregates. By weighing the sample before and after washing out the cement paste, the cement paste content can be estimated. The aggregates collected after the cleaning period are then dried and sieved and their size distribution is analyzed. Because the cement paste is washed out and determined as a whole, there is no provision to determine the dispersion of the mineral admixtures or very fine fillers. As demands for higher performance concretes grow, more precise methods will be needed, such as microscopic observations by scanning electron microscope (SEM), to measure the distribution of the mineral admixtures.D/2 and D, with D the maximum aggregate size) and air content. Several samples (the number is not specified) are taken from the mixer or from the concrete discharge, and the above parameters are measured. The average of all the measurements collected for each parameter and the standard deviation are calculated. The coefficient of variation  gives a measure of the homogeneity of the concrete produced, i.e., a smaller COV implies a more uniform mixture. COV requested. The COV does not depend on the type of concrete selected because it only depends on the relative variation of the parameters for a concrete. This method, proposed by RILEM, is the only attempt by any organization to standardize the process of measuring the efficiency of a concrete mixer.Based on the concept that measuring compositional homogeneity of a mixture can provide evidence of the efficiency of the mixer, RILEM  tried todistribution of cement content, fine aggregates and coarse aggregates in the mixer, measured as described in Sec. 4.2.;variations in compressive strength;variations in consistency as measured by the slump test with increased mixing time.The hybrid method to determine the efficiency of a mixer combines the methods described in Secs. 4.1 and 4.2. The only reference to a hybrid method was found in a paper by Peterson , which hAs many parameters can affect the variations in concrete performance, the method adopted by Peterson was suggested to compare mixers using the same concrete. Peterson gives three types of concrete to select from see . These cAnother indicator of the efficiency of specified mixer is the output rate. The output rate is the amount of concrete produced per a time interval. The output rate is not a measure of the homogeneity of the concrete produced. The output rate depends on the time needed to load the mixer, the mixing time, the discharge time, and the cleaning time, if it is a batch mixer. Very often this last stage is not considered, i.e., cleaning is not considered part of the mixing cycle. This omission is reasonable if the mixer is continuous or if it gets cleaned only once a day. Of course, for reasons of economics, the output rate should be high. However, it should be understood that it is dangerous to base the efficiency of a mixer solely on the output rate because there is no consideration of the quality of the concrete produced.The mixing energy is defined as the product of the average power consumption during the whole mixing cycle and the duration of the mixing cycle. For reasons of economics, the mixing energy should be kept low but the quality of the concrete should be considered first.Johansson  varied tThe power consumption is often used to estimate the workability of the concrete. The theory behind this usage is based on principles of operation of a rheometer. A rheometer is an instrument that measures the stress generated by the material tested while applying a strain. In this case the strain is the constant speed of the blades and the stress is measured by the energy consumption. If it were possible to rotate the blades at different speeds and measure the power consumption at each speed, the mixer could be used to characterize the concrete’s rheological behavior. Nevertheless, while the data obtained will not allow calculation of the rheological parameters of the concrete in fundamental units because the flow of concrete in a mixer is not linear and no equations are available for such a case, the measure of the energy consumption at one speed can be used to compare concretes prepared with the same mixer , or to mTherefore, the mixing energy is a very useful tool to determine variation in the workability of the concrete being produced. However, there is no strong evidence that mixing energy can be used to determine the efficiency of a mixer, unless the only performance requirement is the workability.In determining mixer efficiency, the main focus has been determining the homogeneity and the quality of the concrete produced. It was assumed that the mixer was operating as designed by its manufacturer. But long usage of a mixer leads to wear of the blades and/or scraper, or the build-up of materials (hardened mortar or cement paste) on the blades, the container, and/or the scraper. Wear and build-up will change the geometry of the mixer and therefore the flow pattern of the concrete, and may lead to changes in the concrete produced . To avoiease of cleaning;cost and difficulty of replacing the blades or parts;sensitivity of the mixer to wear and tear of the blades.It can be argued that criteria for a mixer selection should includeMixing is a complicated process that is affected by the type of mixer, the mixing cycle as defined by the duration, the loading method, and the energy of mixing. There are two main types of mixers: batch and continuous. In each type there are several configurations.The efficiency of a mixer is determined by the homogeneity of the concrete produced. It could also be considered as being determined by the energy used in producing a given quantity of concrete of the required homogeneity. This homogeneity is either measured by the composition of the concrete or by the variation of the macroscopic properties, such as compressive strength and workability. It is not clear that the variation of macroscopic properties is very sensitive to variation of composition or to inhomogeneity in the concrete produced. Therefore, a direct measure of the homogeneity of the concrete produced should be the most reliable method for characterizing a mixer. A direct measurement of homogeneity relies on the determination of the concrete composition, such as distribution of the various constituents, including air content, present in various samples taken during the concrete discharge. This composition method was recommended for standardization by RILEM .The mixing energy is the product of the power consumption and the duration of the mixing cycle. It is not necessarily a measure of the quality of a mixer but is used for monitoring workability during mixing, avoiding the necessity of discharging the concrete to measure slump .The literature does not report problems with the mixers commercially available today. The main innovations that are currently being worked on relate to producing mixers that reduce energy consumption and the time of mixing without affecting the quality of the concrete produced.