The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include 2O2. Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05.The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or HhMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O2 toxicity did lead to a reduced endogenous expression of hNTH in A549 cells.Cell killing assays demonstrated cells over-expressing hMYH in A549 cells exposed to O2 and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O2 and its metabolites.Increased expression of the DNA glycosylase repair enzyme  At normal partial pressure, 95% of oxygen molecules (O2) are reduced to H2O and 5% are partially reduced to toxic byproducts by normal metabolism in the mitochondria [2-), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH) all of which make up what are known as Reactive Oxygen Species (ROS) [Oxidative stress leading to the overproduction of free radicals in the lungs is present in many clinical situations. Such clinical settings include acute respiratory distress syndrome (ARDS), infants of prematurity going on to develop bronchopulmonary dysplasia (BPD), pathogenesis of chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, ischemia-reperfusion injury, drug-induced lung toxicity, cancer and aging -4. Althoes (ROS) . ExposurROS are known to attack the lipids, proteins, and nucleic acids of cells and tissues ,7. Lipid2O2 directly to H2O without the production of the hydroxyl radical, and glutathione peroxidase which directly reduces H2O2 and lipid peroxides. Free radical scavengers, which stop free radical chain reactions by accepting electrons, include α-tocopheral (vitamin E), ascorbic acid (vitamin C), niacin (vitamin B), riboflavin (vitamin B2), vitamin A, and ceruloplasmin [Several defense mechanisms exist to combat the damaging effects of ROS. Intracellular enzymatic systems include superoxide dismutase which eliminates the superoxide anion, catalase which catalyzes the reduction of Hoplasmin ,2,9. Theoplasmin ,10.The defense mechanism of interest in this paper involves the repair of oxidative damage through the human DNA base excision repair pathway (BER). BER is the most important cellular protection mechanism that removes oxidative DNA damage . DamagedhOgg1), human Mut Y homologue (hMYH), human Mut T homologue (hMTH), and endonuclease III (hNTH) all of which are present in human cells and involved in the protection of DNA from oxidative damage. The repair enzyme hOgg1 is a purine oxidation glycosylase that recognizes and excise 8-oxoguanine lesions (GO) paired with cytosine. GO can pair with both cytosine and adenine during DNA replication [hMYH is an 8-oxoguanine mismatch glycosylase that removes adenines misincorporated opposite 8-oxoG lesions that arise through DNA replication errors [hMTH hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-GTP, 8-oxo-dATP, and 2-hydroxy-dATP, effectively removing them from the nucleotide pool and preventing their incorporation into DNA  is a pyrimidine oxidation and hydration glycosylase that recognizes a wide range of damaged pyrimidines [hNTH has also been shown to have a similar DNA glycosylase/AP lyase activity that can remove 8-oxoG from 8-oxoG/G, 8-oxoG/A, and 8-oxoG/C mispairs [hNTH are identical to those following hOgg1 , was purchased from ATCC Cat No CCL-185. The cells were grown in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco, Grand Island, NY). Passaging of cells was performed every 3–4 days with cells grown to 80% confluency in a 10 cm cell culture dish (Corning Incorporated, Corning, NY). Cells were kept at 37°C in a humidified, 5% CO2 incubator.The retroviral vector pSF91.1, a gift from Dr. C. Baum from the University of Hamburg in Germany, was constructed with an internal ribosome entry site (IRES) upstream to the gene expressing enhanced green fluorescent protein (EGFP) as previously described .Four DNA repair genes were individually ligated into the retroviral vector pSF91.1.hOgg1 was subcloned into digested plasmid vector pSF91.1, with T4 DNA ligase. DNA sequencing was performed to confirm integrity of the hOgg1 gene.was initially amplified by PCR by primers to introduce a kozak sequence at the 5' end . DigestihMYH was a gift from Dr. A. McCullough  and hMTH was cloned in Dr. Kelley's lab. Plasmid DNA was prepared as above by digestion with EcoRI and SalI and ligated into pSF91.1 as above and sequencing was performed to confirm integrity of the genes.hNTH1-wt fragment was ligated into the empty plasmid vector PUC18. The hNTH1-wt fragment was then excised with both sides flanked by EcoRI restriction sites and ligated into pSF91.1. Proper orientation of the gene was confirmed and sequencing was performed to determine the integrity of the gene.this gene was a gift from Dr. S. Mitra . Digestion with BamHI and SalI was performed and the 5 to 1.2 × 106 particles/ml [DH5α competent cells (Life Technologies, Gaithersburg, MD) with each of the five DNA repair genes were grown in LB-broth with ampicillin (Sigma, St. Louis, MO). Plasmid DNA was prepared and used to transfect phoenix amphotropic cells, from the Nolan Lab , grown to ~80% confluency. On the second day sodium butyrate was added to each plate and incubated at 37°C for 6 hours. Fresh DMEM supplemented with FBS and penicillin/streptomycin was added and the plates were incubated at 33°C. Viral supernatant was collected 24 and 48 hrs later, filtered through a 0.45 μm acrodisc syringe filter  and frozen at -80°C for later use. Retroviral titers were determined by fluorescent-activated cell sorter (FACS) analysis. Titers of viral supernatant were 8 × 10icles/ml .5 A549 cells were suspended with the viral supernatant and plated in 1 well of a 6-well plate along with polybrene (Sigma, St. Louis, MO). This exposure was performed 6 hours per day for three days. At approximately five days from the beginning of the infection, the infected cells were analyzed using flow cytometry and sorted for EGFP expression.2.5 × 10hOgg1 , hMTH , hMYH (Oncogene Research Products, Darmstadt, Germany) and hNTH (Proteintech Group Inc, Chicago, IL) all at a dilution of 1:1000 except hNTH which was diluted 1:2500. They were then washed 2 times with TBST and 2 times with 0.5% blocking solution, 10 minutes per wash. The membranes were incubated with anti-rabbit secondary antibodies at 1:1000 for 1 hour at room temperature. Lastly, the membranes were washed 4 times with TBST, 15 minutes per wash. The membranes were briefly soaked in BM chemiluminescence blotting substrate (Roche Diagnostics, Indianapolis, IN) and then exposed to high performance autoradiography film (Amersham Biosciences, Buckinghamshire, England). Kodak Digital Science 1D Image Analysis software was utilized to quantify the region of interest (ROI) band mass of individual bands on films where visualized differences were detected.Cell pellets of sorted cells were resuspended in NuPage buffer (Invitrogen, Carlsbad, CA) and protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). 20 ug of protein were loaded into individual lanes of a NuPage Bis-Tris Gel (Invitrogen, Carlsbad, CA). The gel was then transferred to nitrocellulose paper (Osmonics Inc, Gloucester, MA). The membranes were then blocked with 1% blocking solution (Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibodies to 2 and 5% CO2. Individual plates were removed after 12, 24, 48, and 72 hours of exposure. Control A549 cells were incubated in a normal 37°C humidified-5% CO2 incubator. O2 concentrations were monitored with a MAXO2 analyzer . Four days from the beginning of the exposure, cells were assessed for cell growth/survival using the sulforhodamine B assay (SRB assay).Sorted EGFP positive A549 cells infected with the above DNA repair genes were counted and seeded into 96-well plates at a density of 1000 cells/well, 6 wells per gene. Six hours after seeding, individual plates were placed into an oxygen chamber supplied by Dr. L. Haneline (Wells Center for Research, Indianapolis, IN) located in a 37°C incubator. The oxygen chamber was then infused with 95% OThe SRB assay (Sigma, St. Louis, MO), developed by the National Cancer Institute, provides a sensitive measure of drug-induced cytotoxicity through a colorimetric endpoint that is non-destructive, indefinitely stable, and visible to the naked eye. This assay was used to assess the cell growth/survival of over-expressed cells . Cold 102O2 (Sigma, St. Louis, MO). All plates including control plates were then placed into a 37°C humidified-5% CO2 incubator. Four days after exposure, cells were fixed and assessed for cell growth/survival by the SRB assay.Sorted EGFP positive A549 cells were seeded into 96-well plates at a density of 1000 cells/well. Six hours after seeding, individual plates were then exposed to radiation at doses of 250, 500, 1000, and 1500 Rads or 0.2 mM, 0.4 mM, and 0.6 mM H2 incubator. Every 24 hours for 4 days, 1 plate was removed and the cells were fixed and analyzed by the SRB assay looking at cell growth under non-toxic conditions. Growth curves and exponential growth equations were determined to look at the doubling time (DT) of cells infected with each repair gene of interest compared to vector infected and uninfected wild type cells.Sorted EGFP positive A549 cells and wild type cells were seeded individually onto four 96-well plates at 1000 cells/well. All the plates were placed into a 37°C humidified-5% COt test with significance being accepted for p < 0.05.All drug exposure experiments were performed at least three times and individual drug doses included 6–8 wells for each group of infected cells. Analysis of cell growth and exponential growth equations were determined using Microsoft Excel. All experiments involving drug exposures were normalized to the zero dose. Data are expressed as means ± SE. The significance of differences were calculated using the paired Student's hOgg1, hMYH, hMTH, and hNTH were ligated into the retroviral vector pSF91.1 .Western blot analysis was performed on sorted cells in order to verify over-expression of the four genes of interest. 2 and 1000 Rad). Cells over-expressing the repair genes hOgg1, hMYH, hMTH, and hNTH did not lead to altered expression of the other endogenous repair genes under the above conditions when compared to each other or pSF91.1 vector control cells  at 24 and 48 hrs after the onset of exposure. Endogenous hMYH and hMTH were expressed to the same degree. hOgg1's endogenous expression was below the level of detection using western analysis (results not shown). When analyzing endogenous hNTH expression, it was noted that hyperoxia at 24 hrs and 48 hrs resulted in reduced protein expression by 93% and 64% respectively. There also was a small increase in expression of hNTH noted after 1000 Rad one day post exposure that was back to baseline by two days post exposure. ROI band mass quantification demonstrated this finding  to detect improvements in survival [hMYH and its impact on survival. Another study has investigated the repair function of hMYH in MYH-deficient murine cells. It was demonstrated that transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:GO repair [The primary agent utilized to induce the formation of ROS was an oxygen rich environment. The use of oxygen as a stressor leading to the formation of ROS, offers a distinct advantage over IR and Hsurvival . AdditioO repair .hMYH clearly grow at a slower rate when compared with the other enzymes. The mechanism behind this is not understood at this point in time. The repair action of hMYH is known to remove adenines misincorporated opposite 8-oxoG lesions. This lesion occurs when a C/GO lesion is allowed to replicate before being corrected by hOgg1. Repair by hMYH is not a final corrective measure. The product of hMYH activity is the lesion C/GO, which allows hOgg1 to have another opportunity to remove 8-oxoG opposite cytosine. We know that A549 cells possess the hOgg1 gene based on a previous study demonstrating the presence of this gene after amplification by genomic PCR [hOgg1 in A549 cells by using an 8-oxoguanine bioactivity assay. Therefore, our explanation of these results is that the slowed growth created by hMYH may provide a wider window of opportunity for the repair process to take place, which ultimately grants endogenous hOgg1 another opportunity to remove the 8-oxoG lesion created by oxidative stress.An interesting observation noted while doing our experiments lead us to look at individual growth characteristics of cells over-expressing each of the oxidative repair enzymes. Cells over-expressing the repair enzyme omic PCR . We alsoAs noted in the methods section, the SRB assay provides a sensitive measure of drug-induced cytotoxicity that is used to assess cell proliferation/survival. The reduced cell proliferation of A549 cells over-expressing hMYH under non-toxic conditions may likely underestimate the magnitude of the protective effect of this particular repair enzyme. This may in fact make the results even more significant.hMYH that may predispose to familial colorectal cancer [hMYH variants in lung cancer patients and have not identified any clear pathogenic biallelic hMYH mutations or an over-representation of hMYH polymorphisms [hMYH, but a single nucleotide polymorphism (SNPs) has been noted [hMYH is very important in preventing somatic mutations leading to cancer in the gastrointestinal tract. Although studies to date have not demonstrated this same relationship with lung cancer, we do know that the lungs are subjected to large quantities of ROS under certain conditions as discussed earlier. The formation of mutations from oxidative stress does have other deleterious effects on cells including cellular death by necrosis and apoptosis. Tissue viability is dependent upon mutation correction and replication of the surviving cells to replace those that have died. The ability to enhance cellular survival, after specific oxidative exposures, is evident after increased production of the hMYH repair gene in these experiments.Recent studies have discovered hereditary variations of the glycosylase l cancer ,47. Otheen noted . The imphOgg1 was elevated following exposure to crocidolite asbestos which is known to cause an increase in 8-oxoG levels [hOgg1 protein expression that was not observed with H2O2 [hOgg1 protein expression in rat lung after exposure to cadmium, a known carcinogen associated with the formation of intracellular ROS [hOgg1, hMYH, and hMTH at 24 and 48 hours following exposure. It was noted that endogenous hNTH was reduced after hyperoxia at 24 and 48 hours after the onset of exposure. One would speculate that this reduction in endogenous hNTH secondary to hyperoxia is related to either decreased production or increased destruction in response to O2 exposure. Over-expression of this repair enzyme did not result in improvements in survival after O2 exposure based on our experiments. It may be that endogenous levels are adequate to correct this specific mutational burden for these experiments.We additionally wanted to determine the level of endogenous expression of the glycosylase repair genes in the pulmonary epithelial A549 cell line. Others have demonstrated how different stressors lead to alterations in the endogenous production of specific repair genes. For example, it has been shown that endogenous gene expression of G levels . It has ular ROS . In our Furthermore, no previous studies have determined how cells over-expressing specific repair genes may impact endogenous expression of the other oxidative BER genes under both normal and oxidative stress conditions. We were also able to demonstrate that endogenous expression of glycosylase repair genes were not altered under these conditions secondary to the over-expression of any of these genes. This is an important finding for interpretation of survival data; protection of cells is due to the over-expression of the specific gene and not due to enhancement of other endogenous repair enzyme levels, at least for the genes studied under these conditions.Some limitations may exist in using a lung carcinoma cell-line, which likely differs both in proliferative properties as well as in response to oxidative stress in comparison to primary epithelial cells. The enhanced cell growth observed with cell lines may be more reflective of undifferentiated alveolar type II cells which are likely to replace terminally differentiated alveolar type I cells after injury/death due to oxidative stress. This may not be a true reflection of growth under non-toxic conditions when very little cell division is occurring. This is an inherent problem observed when comparing cell lines with primary cells and results need to be interpreted in a way that considers this.in vivo at this stage. It certainly would appear that the protection observed is modest in degree in this pulmonary epithelial cell line. Further experiments assessing the function of the repair enzyme hMYH in this model will be important to perform in order to delineate the findings of slowed growth under normal conditions and improved survivability under conditions of O2 and IR. More research looking at the potential for combination therapy, including DNA repair mechanisms in conjunction with other antioxidant defense mechanisms may be another approach to enhancing cell survival, which may lead to better clinical outcomes. Alternatively, cell survival may not be the most important end point for hyperoxia studies. Given that 8-oxoG, if left unrepaired, leads to G:C to T:A transversions, there may be an increase in mutational burden by these cells that isn't reflected in cell survival. Further experiments studying the impact on mutation production is underway. Ultimately, experiments need to be done in animal models to determine the translation to in vivo pulmonary cells.It is difficult to know how this will translate to pulmonary epithelial cells hMYH may enhance survival of a pulmonary epithelial cell line after exposure to conditions of IR and hyperoxia. We have also demonstrated that over-expression of hMYH leads to a slowing of growth of A549 cells under non-toxic conditions, which may in part play a role in this enhancement of survival by providing a wider window of opportunity for repair of oxidized lesions to occur. Lastly, we demonstrated that over-expression does not lead to altered endogenous expression of these repair genes. As the understanding of DNA repair mechanisms continues to grow and the evolution of gene therapy takes place, more treatment options may be available in the clinical setting to help with many disease processes including the damaging effects of oxygen and its metabolites.In summary, we have demonstrated that over-expression of the DNA glycosylase repair enzyme 2O2; ionizing radiation, IR; internal ribosomal entry site, IRES; long terminal repeat, LTR; oxygen, O2; Sulforhodamine B, SRB; reactive oxygen species, ROS; region of interest, ROI; Tris-Borate-EDTA, TBE; tris-buffered saline Tween-20, TBST; 8-oxoguanine, GO and 8-oxoGapurinic, AP; base excision repair, BER; Dulbecco's modified Eagle's medium, DMEM; deoxyribose phosphate, dRP; enhanced green fluorescent protein, EGFP; fetal bovine serum, FBS; hydrogen peroxide, HTK conducted the majority of the research experiments, performed the statistical analysis, and drafted the manuscript. MR conducted some of the cell survival experiments and participated in the design of the study. YX and XC helped with production of the lesion specific DNA repair genes. MK conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps  may not consistently kill MTB organisms.In the field of clinical mycobacteriology, n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks.An inclusive viability study (n = 18), resulting in 0% viability.Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again  or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures. Mycobacterium tuberculosis (MTB), the causative organism of tuberculosis, has the distinction of repeatedly being ranked within the top five most commonly laboratory-acquired infections (LAIs) [M. tuberculosis and other pathogens as the infectious agent [s (LAIs) -3. In 19us agent . With thus agent . A more us agent , in spitus agent .Due to the nature of this organism, containment level three (CL3) laboratory operational and physical requirements have been recommended for manipulation of the live organism in North America . TherefoCurrently, the application of molecular methodologies for rapid diagnostics of MTB, such as nucleic -acid amplification based identification and subtyping schemes, in addition to extensive genomic and proteomic research in this area, necessitates the removal of material derived from this organism out of a CL3 laboratory to perform the work in a less restrictive containment level 2 (CL2) rated area. Commonly, due to both limited CL3 space, costs and preventative maintenance needs, high-tech equipment such as liquid handling robots and sequencers are shared and can be housed in a central "DNA core" CL2 laboratory.M. tuberculosis. The existing literature is limited in regards to viability testing of material derived from MTB with respect to safe manipulation outside of a Biological Safety Cabinet (BSC). To date, no conclusive study has confirmed that this organism is noninfectious after theoretical 'deactivation' steps. A few reports concerning survival after different heating kill treatments for DNA extractions [To consider the biosafety impact of removing organism material from a CL3 and manipulating it in a CL2, part of a risk assessment undertaken included the review of current literature on decontamination verification and viability testing of ractions -9, heat ractions ,11 and cractions  were fouSince, by definition, a risk assessment is based on, but not limited to, the properties of the agent used, personal risk factors, manipulation techniques, and the training and experience of staff, it is necessary to develop a method encompassing these factors to validate the safe removal of material derived from MTB from a CL3 laboratory for use in other laboratory areas. This approach was applied to all our methodologies and took into consideration variables such as interpersonal technique, culture load, temperature fluctuations and statistical significance.M. tuberculosis complex, Accuprobe (GenProbe, San Diego, CA). Methods that resulted in viable MTB were revised and retested. All work completed was performed in a CL3 environment, using Class II Type B2 biosafety cabinets (BSCs), and personal protective equipment (PPE) including N100 particulate respirators, double gloves, and protective gowns. The following describe the protocols used in our laboratory at the start of this study.The current viability study consisted of the evaluation of a total of 226 material extracts, consisting of 208 initial extracts and 18 extracts tested after revision of faulty protocols .A total of 45 lysates used for PCR testing were tested for viability. Thirty-six lysates were prepared from actively growing cultures by three technicians that normally perform the testing procedure. Briefly, a loopful of culture is placed in a pyrex glass bottle containing 1 mL distilled water and glass beads and vortexed in a BSC. Generally this suspension has a turbidity of > 1 McFarland. This suspension is transferred to a screw-capped vial and is placed in a boiling water bath for 10 minutes followed by transfer into a tube containing 0.5 mm silica beads and mechanically lysed for 2 minutes with a Mini-8-Beadbeater (BioSpec Products, Bartlesville, OK). The resulting lysate is spun down for removal of debris and the supernatant transferred to a new tube to be used for PCR. Lysates were processed in duplicate and the position in water bath was noted (i.e. periphery or centre). A proportion of lysates (100 uL) were planted pre and post bead-beating. An additional nine previously frozen lysates stored at -20°C were also inoculated into 12B media (n = 12 slides). The heat fixed slide material was emulsified using a sterile swab and sterile distilled water. The suspension was transferred to a pyrex glass bottle containing 1 mL sterile water with beads, vortexed and 100 μl was inoculated into a 12B media vial.Slides were prepared in duplicate according to our standard protocol: a loopful of organism is suspended in water with beads, vortexed and one drop added to a glass slide containing one drop of 0.5% phenol serum (made in-house). The slides were allowed to dry and were placed on a 95°C slide-warmer (Lab-Line Instruments, Melrose Park, IL) for 15, 30, 45 minutes, 1, 1.5 and 2 hours (M. tuberculosis organism were processed as follows. A volume of 25 mL of Middlebrook 7H9 broth was inoculated and incubated at 37°C for 20 days. The culture was centrifuged at 1900 × g for 15 minutes at room temperature and the pellet was resuspended with 2 ml of ice-cold phosphate buffer solution (PBS) pH 6.8 supplemented with 1% Tween-80. The sample was centrifuged as before at 4°C and the pellet washed twice in ice-cold 1% Tween-80 in PBS. Silicon beads (0.5 mm) and 500μl lysis buffer (2% CHAPS, 2% Triton X-100, 9.5 M urea and 1% DTT/TBP in water) were added to the pellet. The sample was mechanically lysed for 30 seconds and cooled on ice for 30 seconds, repeated eight times. The sample was then filter sterilized with a PES membrane, 0.22 μm Millipore filter unit (Millipore, Etobicoke, ON) into a 2 ml microcentrifuge tube on ice and stored at -80°C.Twenty-six extractions of M. tuberculosis . These 12B culture vials were confirmed positive for et al. [Two of 25 vials tested for viability from protein extractions became positive. Confirmation of viable MTB in these test vials was confirmed as described above. Based on these results, the methods for slide fixation and protein extraction were modified. Slides were fixed prior to staining using 5% phenol in ethanol according to Chedore et al. , and proM. tuberculosis culture non-viable [et al. was prompted by a case of pulmonary tuberculosis acquired by a laboratory technician performing the standard method for IS6110-RFLP in a CL3 mycobacteriology [There are few publications that review the efficacy of methodologies that render material extracts from a n-viable -10. One eriology . In thiseriology .et al. also found that submerging the culture at 100°C for 5 minutes rendered the sample completely non-infectious; data that was supported by work previously done in 1994 by Zwadyk et al. [et al. showed that using a 95°C heat block failed to completely inactivate the culture, and in fact, using an internal temperature probe demonstrated that the tube did not reach the intended temperature even after 20 minutes [Bemer-Melchior k et al. . Althoug minutes . Another minutes . Again,  minutes . In prepet al for the purpose of RFLP typing [et al., resulted from incompletely submerging sample tubes or cell density within the tube [Genomic extractions, the most commonly performed test in our laboratory, are conducted using the standardized method outlined in 1997 by Van Embden P typing . Prior vthe tube , and to M. tuberculosis. Further sampling of the DNA extractions performed in our laboratory (10% of each lysate batch) is ongoing for quality assurance purposes, i.e. to confirm integrity of reagents as well as to monitor staff performance and adherence to protocol. In addition, continuous sampling attaches statistical significance to the claim that this method ensures that the extracted material is 100% non-viable and guarantees staff safety.To both preserve the integrity of genomic DNA for the use of fingerprinting and allow the sample to be further processed safely outside a CL3 laboratory, it is recommended to complete DNA extractions with CTAB as per the standard protocol to confirm the complete inactivation of et al. concluded that inactivating mycobacteria by heat lysing at a temperature of 100°C for 30 minutes did not inhibit its ability to be amplified by PCR or strand displacement amplification [For procedures that do not require intact, high quality DNA such as PCR testing, our laboratory depends on the much more expedient lysate method of boiling culture at 100°C for 10 minutes, followed by mechanical lysis for 2 minutes to release DNA. Although crude, this procedure is adequate for our PCR testing needs, has been shown to completely destroy live organism in our laboratory, and is consistent with other studies ,8. The sThe viability testing of the two methods outlined above for DNA extractions were performed by various technicians who routinely follow these procedures, lending interpersonal variability to the study. It was demonstrated that the small nuances to procedure, such as the varying density of culture used, did not affect the method employed to deactivate the organism.The last routine test assessed for organism viability was the slide preparation. While phenolics are known to be tuberculocidal, the use of phenol serum alone or in conjunction with heat in slide preparation is not sufficient for killing of the live organism. Flaming of the slide was not an option due to facility requirements that prohibit open flames inside a BSC, despite this, flaming of smear material was found in prior studies to unsuccessfully inactivate smear material . The priExamining laboratory protocols for staff and building safety should be an integral part of any CL3 laboratory program. Implementation of a policy to test all procedures used routinely for viability in addition to new methodologies is highly recommended. As an example, applying this policy to a new procedure being utilized in the research arm of our laboratory proved valid: a protein extraction method being developed initially showed that of 2/26 tested samples yielded viable MTB. The researcher concluded that it was due to a clogging of the filter from large particles in the lysed culture used to isolate the proteins and revised the protocol to centrifuge the mixture after final lysis steps to remove cellular debris before filtration. This has proven successful so far, and continuous sampling of extracts is ongoing before removing the material from the CL3. To date, all have been negative.It is imperative to evaluate and record the actual rate of viability of DNA lysates from deactivated MTB cultures in individual laboratory settings . This inThe author(s) declare that they have no competing interests.KB carried out the studies involving genomic DNA extractions and drafted original and final manuscripts. TB tested all smear preparations, compiled viability data and assisted with the writing of the manuscript. MS contributed by testing protein extractions and writing the method used. CT contributed by testing of crude lysates and editing of manuscript. MS contributed by testing protein extractions and writing the method used. AK and JW conceived of the study, and participated in its design and ongoing coordination. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


Desmoplastic infantile ganglioglioma (DIG) is a rare WHO Grade I tumor of infancy that is characterized by large volume, superficial location, invariable supratentoriality, fronto-parietal lobe predilection and morphologically, by an admixture of astroglial and neuroepithelial elements in a desmoplastic milieu. With over 50 cases described, the histologic and radiographic spectrum of DIG has been well-characterized. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology; however, the cytologic features of this rare tumor have only been reported once previously.We present herein cytomorphologic findings from the intraoperative aspiration of a typical case of DIG diagnosed in a 1-year-old male. As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed  large neuronal cells with eccentrically located hyperchromatic nuclei  and abundant unipolar cytoplasm. Rare smaller astroglial cells were intermixed. Despite the tumor's characteristic desmoplastic histologic appearance, no stromal fragments were identified on the aspiration material.A differential diagnosis is presented and analyzed in detail and it is concluded that when these large neuronal cells are encountered in an aspirate of a brain mass in a child, a combination of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities. They mo in 1987 . DIGs ar in 1987 . The sup in 1987 . To contA one-year-old boy was noted to have a striking increase in head circumference as compared to a previous measurement. Neurological examination and developmental status were normal at that point. Within 2 weeks, the patient deteriorated rapidly, with poor mobilization, feeding and verbalization. He was brought to the emergency room where an emergent computed tomographic scan showed a large left hemispheric cerebral mass Figure  with an ® 2000 Automated Slide Processor (Cytyc, Boxborough, MA) according to the manufacturer's instructions. For the tumor specimen, approximately 10 × 6.5 cm of fragmented gray and white cerebral tissue was received and entirely processed routinely: tissue sections were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned to 4 μ-thick sections and stained with hematoxylin and eosin, Nissl stain and reticulin. The immunohistochemical profile of the tumor was evaluated on 4 μ thick, formalin-fixed, deparaffinized sections using a DAKO Autostainer (Carpinteria, CA, USA) based on the avidin-biotin-peroxidase complex with antibodies ki-67 (dilution 1:320, DakoCytomation Corp, Carpinteria, CA), synaptophysin (dilution 1:600, DakoCytomation) and glial fibrillary acid protein [GFAP] (dilution 1:10, DakoCytomation).For cytology, a slide was prepared from 50–60 cc of straw-colored fluid utilizing the ThinPrepAs evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed  large neuronal cells (~70 μm diameter each) with round eccentrically placed uniform hyperchromatic nuclei, undulating and slightly convoluted nuclear membranes, and abundant unipolar granular cytoplasm (ganglion cells) figures  and 1-3.Vascular structures or stroma were not present. Histologically, the tumor was partially attached to the dura and was present in the subarachnoid space. The bulk of the specimen was a variably cellular desmoplastic component whose predominant constituent cells were elongated spindle cells arranged in a reticulin-rich, storiform pattern figure . At highThe clinical presentation, radiographic appearance and histopathologic features of this case are entirely consistent with those described for desmoplastic infantile ganglioglioma -3. ThereThe distinctive clinical features of DIG, being a typically superficially located tumor occurring in young children , may render them particularly amenable to pre-operative assessment using aspiration cytology. In addition, familiarization of practitioners with the cytopathologic features of DIG may be useful because 1) With the aforementioned cases of DIG regressing after subtotal resection ,8, it miThe finding of large cells with eccentrically located nuclei and abundant unipolar cytoplasm in an aspiration specimen of a cerebral mass occurring in a young person should generate a differential diagnosis that includes DIG, atypical teratoid/rhabdoid tumor (AT/RT), dysembroplastic neuroepithelial tumor (DNT), ganglioglioma, supratentorial primitive neuroectodermal tumour (PNET) with ganglionic differentiation (ganglioneuroblastoma), anaplastic large cell lymphoma and pleomorphic xanthoastrocytoma. In our opinion, clinical features as well as immunohistochemical analysis can significantly help reduce the likelihood for most of the aforementioned entities. The distinction of DIG from AT/RT is probably of the greatest prognostic significance, since in contrast to DIG, AT/RT is a highly malignant tumor that is almost uniformly fatal . AlthougImmunohistochemically, the rhabdoid cells of AT/RT co-express vimentin and epithelial membrane antigen , in contMorphologically, the distinct cytoplasmic borders, "inclusion-like" cytoplasmic globule and overall dense eosinophilia of the cytoplasm of rhabdoid cells, in conjunction with the aforementioned clinicopathologic parameters, may help in their distinction from ganglion cells of DIG ,10. The Other less likely differential considerations include pleomorphic xanthoastrocytoma and anaplastic large cell lymphoma (ALCL), both of which may contain large cells with eccentrically located nuclei and abundant cytoplasm. The temporal lobe predilection, lack of neuronal differentiation, presence of xanthomatous cells, smaller tumor size and older age of patients with pleomorphic xanthoastrocytoma should permit an easy distinction of the large cells in this tumor from those of DIG. Primary brain ALCL is exceedingly rare and generally occurs in older individuals, with a mean age of 29 years in one series . ImmunorWhen the cytomorphologic findings of DIG are described in more cases, it is likely that the cytologic spectrum will mirror the histologic heterogeneity of this tumor. For example, astroglial cells predominated in the two cases of Hasegawa et al  while thThe author(s) declare that they have no competing interests.All authors made substantial contributions to the intellectual content and/or presentation of the manuscript. ITO (cytologist), diagnosed the cytopathological aspects of the case and co-supervised the entire project. JHK (neuropathologist), diagnosed the histological aspects of the case and co-supervised the project. OF wrote the initial version of the manuscript. MRM, DH and AWZ collected pathological, clinical and/or photographical information and revised the manuscript.


Nicotiana attenuata, is found primarily in large ephemeral populations  after fires in sagebrush and pinyon-juniper ecosystems and in small persistent populations (for many growing seasons) in isolated washes typically along roadsides throughout the Great Basin Desert of the SW USA. This distribution pattern is due to its unusual germination behavior. Ephemeral populations are produced by the germination of dormant seeds from long-lived seed banks which are stimulated to germinate by a combination of unidentified positive cues found in wood smoke and the removal of inhibitors leached from the unburned litter of the dominant vegetation. Persistent populations may result where these inhibitors do not exist, as in washes or along disturbed roadsides. To determine if this germination behavior has influenced population structure, we conducted an AFLP , ISSR  and ISSR+ AFLP  analysis on plants originating from seed collected from populations growing in 11 wash and burns over 11 years from the SW USA.The native annual tobacco, Genetic variance as measured by both ISSR and AFLP markers was low among sites and comparatively higher within populations. Cluster analysis of the Utah samples with samples collected from Arizona, California, and Oregon as out-groups also did not reveal patterns. AMOVA analysis of the combined AFLP and ISSR data sets yielded significantly low genetic differentiation among sites (Φct), moderate among populations within sites (Φsc) and higher genetic differentiation within populations (Φst).We conclude that the seed dormancy of this post-fire annual and its resulting age structure in conjunction with natural selection processes are responsible for significantly low among sites and comparatively high within-population genetic variation observed in this species. Nicotiana attenuata Torr. ex Watson (Solanaceae) (synonymous with N. torreyana Nelson and Macbr.) is an annual native to the Great Basin Desert of California, Nevada, Idaho, and Utah (USA) [N. attenuata seeds are stimulated to germinate by unidentified factors in wood smoke [N. attenuata produce seeds that vary in their genetically-determined primary dormancy [ah (USA) -3 and prah (USA) ,4-9. Posah (USA)  can accood smoke  which leN. attenuata are self-compatible and outcrossing does not significantly affect seed production, seed mass or viability [This well-characterized germination behavior likely affects the genetic structure of this potential annual. Genetic structure of a population results from mutations, gene flow , drift, and selection, all acting in the context of an organism's life history traits . Geneticiability  indicatiN. attenuata plants from wash and burn populations in the SW Utah  Are plants growing in burn and wash populations genetically distinct? 2) Are plants growing in the same washes genetically similar through different years? 3) What is the genetic makeup of plants found growing across large burns and geographically adjacent populations? While genetic diversity among the various Nicotiana species has been studied with RAPD [Nicotiana species.Specifically, we compare plants growing from seeds collected from 11 large populations after fires, from small populations in 10 washes, from plants in transects across 5 large burns, and from plants growing in specific areas over 10 years during which a small wash population erupted into a large burn population as a result of a fire and returned to become a small wash population. By analyzing the genetic diversity across these ith RAPD  and AFLPith RAPD  markers,ith RAPD , this isprinciple co-ordinate (PCO) analyses. The Jaccard similarity index [unweighted pair group method average (UPGMA) dendrogram revealed a lack of distinct spatial or temporal structure and had brush- or star-structures with nodal bootstrap values of less than 60% (data not shown). The samples collected from the greatest spatial distances, namely California, Oregon and Arizona, did not form separate clusters from any of the Utah populations. A cluster analysis of 10 wash and 5 burn populations all grown from seed collected in 1999  and plants from Pahcoon, the highest (0.2043 ± 0.0027) (Table A Bayesian approach  was usedest 0.204 ± 0.0027p < 0.05) revealed highly significant differences at p < 0.001. AFLP and ISSR data was combined for an AMOVA analysis of all analyzed Utah populations. From this analysis, all three Φ categories were highly significant  . The Φst values from the AFLP, ISSR and the combined data sets were separately correlated with geographical distance and all revealed non significant correlations The analysis revealed high levels of heterozygosity, with total heterozygosity from all populations (0.2771 ± 0.0018) being higher than that from comparable analyses of self-pollinating annual plants 0.131) . The ISS1 . The I2 of each other. Pahcoon, was the site from which highest number of populations were analyzed. (9 populations), whereas, from other sites smaller numbers of populations were analyzed.The principle conclusion of this study is that large amount of genetic variation measured by AMOVA, within populations at a particular area significantly dwarfed that observed among sites, among populations growing in burns or washes, or collected during subsequent years growing at a given site. The conclusion that the genetic variation between neighbors is greater than that found between temporally- or spatially-separated populations, is dramatically reflected in the plants sampled along transects through the Pahcoon Springs burn. Only a small fraction (12.6%) of the total genetic variance is found among the 8 sub-samples from the extreme corners of this burn that covered more than 5000 hectares , while tPlatanthera leucophaea, Holsinger et. al [B is substantially larger than the estimates from the AMOVA analyses (θB = 0.392 Vs Φst = 0,252). In our study, AFLP + ISSR analysis also showed such difference (θB = 0.3305 Vs Φst = 0.156) whereas, exactly opposite values were found separate analysis of AFLP and ISSR data sets .F-statistic was estimated by using Bayesian approach with an analogue of the F-statistic, the Φ-statistic. Low but significant genetic differentiation was estimated among sites (Φct) from AFLP (0.027), ISSR 0.093) and AFLP + ISSR (0.046) data sets, whereas within populations, the Φst values were very high . Another major difference from the current study was that G. macleayana showed significant isolation by distance.Dormancy is a major adaptive response of native plants that allows them to cope with environmental variation and provide a means of habitat selection -29. Morenceolata , Callunavulgaris , Clarkiaillensis  and Lesqfendleri  it has bfendleri ,38. Othefendleri . Studiescleayana , which aSeed dormancy increases the effective generation time of this annual plant and by doing so, prevents genetic decay and inhibits the formation of spatial structure between geographically distinct populations . AdditioN. attenuata has all the characteristics of species pollinated by moths at night (white fragrant flowers scenting and becoming receptive at night) but day-active humming birds (Selaphorus sp.) and bumblebees (Bombus sp.) are also known to visit the flowers [Hyles lineata and Manduca species and could be dispersed by wind, water transport and animals, but none of these mechanisms are well documented. The seeds are eaten by various ground squirrels [N. attenuata calyx is sticky and glandular and could be dispersed by adhering to animals. However, the plants ability to produce the defense secondary metabolite nicotine in substantial quantities in its calyx [N. attenuata seeds, burned the sagebrush to promote its growth and are likely to have transported seeds throughout its range in North America [N. attenuata genotypes across the landscape by humans who were smoking this plant may have contributed to the lack of correlation between geographical and pair wise Φst values, as reflected by Mantel test for isolation by distance.Seeds of quirrels  but are quirrels  suggeststs calyx  may be a America . Hence, N. attenuata populations from Utah revealed by AFLP and ISSR analysis is a likely result of combination of random dispersal by humans and its seed dormancy.In summary, we conclude that the unusual nature of the N. attenuata populations in Utah showed: 1) high similarity across collection sites; 2) small difference between populations growing in burns or washes; 3) small differences between growing seasons; and 4) large difference between individuals growing within populations.We conclude that the genetic structure of 2 region of the SW corner of Utah (T38S R10W-T43S R19WUSA). Collections from Arizona (Flagstaff), Oregon (Eugene) and California (Sequoia Natl. Park) served as out-groups. In Utah, seeds were collected from plants growing at 6 locations for a number of years and were used for a time series analysis  were exposed for 1 h to 100 μL liquid smoke (House of Herbs INC., Passaic, NJ, USA): water (1:300, v/v) in 1-mL shell vials and 5 seedlings were planted in soil and grown to the rosette-stage in a glasshouse. Leaves from one plant randomly selected plant from each collection were harvested for DNA extraction.Leaves were flash-frozen in liquid nitrogen, ground to powder and suspended in 750 μL of 100 mM Tris/50 mM EDTA (pH 8.0), containing 250 μg/mL RNase A. Eight μL liquid laundry detergent  were added. After 60 min incubation at 60°C and subsequent addition of 80 μL of 5 M NaCl, the suspension was centrifuged for 5 min at 16,000 × g. The supernatant was removed and extracted with phenol/chloroform. The DNA was precipitated with 600 μL isopropanol, pelleted by centrifugation at 16,000 × g for 5 min, washed with 200 μL 70% ethanol and dissolved in 50 μL of water. The purity and concentration of the extracted DNA were assessed by electrophoresis on a 1% agarose gel and optical density spectrometric measurements. Both AFLP and ISSR procedures were performed on the same DNA samples.Restriction. The enzyme combination EcoRI/MseI was used to restrict 500 ng of genomic DNA per sample. A 10 μL digestion mix (2 μL NEB Buffer #2, 2 μL 10 × BSA, 1.25 μL Mse I, 0.25 μL Eco RI, 4.5 μL water) was added to 10 μL preparation of genomic DNA and incubated at 37°C for 3 h. (2) Ligation of adaptators. The double stranded EcoRI and MseI adaptator sequences designed by Zabeau and Vos [Pre-amplification. Primers designed by Vos [Amplification. Three 6-selective nucleotide EcoRI and MseI primer combinations, also designed by Vos [Nicotiana tabacum by Ren & Timco [The four-step AFLP marker production procedure of Sharbel  was foll and Vos  were ligd by Vos  complemed by Vos . (4) Ampd by Vos  and demo & Timco , were us & Timco . Each of & Timco .The separate PCR amplification products generated by each of the three primer combinations were loaded together with a ROX 500 GeneScan size standard onto the ABI Prism 310 automated genetic analysis system as described in the manufacturer's instructions. The samples were run with the following GeneScan settings: "GS STR POP 4 (1 mL) D" module; 150000 V run Voltage; 5 second sample injection; 60°C gel temperature, and 9 m Watt's laser power. The distinct emission spectra of the three fluorescently labeled Eco RI-primer types made it possible to distinguish the DNA fragments resulting from each of the different primer combinations separately while the samples were being separated in the same electrophoresis capillary.Collection of raw data and size alignment of the AFLP fragments was performed with ABI Prism GeneScan Analysis Software (Applied Biosystems) with the internal standard. Aligned data were subsequently imported into Genographer  for band2) 0.8 U Taq DNA polymerase (Eppendorf) 0.1 mM dNTPs, 0.3 μM primer. After 5 min initial denaturation at 94°C, 45 cycles of 1 min denaturation, 45 s annealing at 50°C, and 2 min for extension at 72°C, were followed by 5 min final extension in the PCR cycling program. A total of 55 primers were screened and 5 primers (Table The PCR reaction 25 μL) contained 20 ng genomic DNA, buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl5 μL contunweighted pair group method average) clustering method and PCO  options of software MVSP  programGenetic diversity was estimated for SET II   as heterozygosity using the Bayesian approach of Holsinger et. al. For thiThe SET II was used to calculate molecular variance from the combined and separate AFLP and ISSR data sets  as partitioned into individual and population components with an AMOVA (ver1.55: ). We alsRB carried out the entire ISSR analysis, the analysis of the AFLP data and contributed to writing the manuscript. DS grew the plants, extracted the DNA and conducted the AFLP analysis. CAP collected seeds, grew the plants and extracted the DNA. ITB was responsible for coordinating the study, collecting seeds for the analysis, and wrote the manuscript.Nicotiana attenuata. Levels of significance are given in the upper diagonal of the matrix: *p < 0.05, **p < 0.01, ***p < 0.001 and NS, Non Significant at p < 0.05. p-value Indicates the probability that a random genetic distance (Φst) is larger than observed distance and are based on 1000 iterations steps.Table 5 Pair-wise genetic difference  among 25 populations of Click here for file


The process by which the Neanderthals were replaced by modern humans between 42,000 and 30,000 before present is still intriguing. Although no Neanderthal mitochondrial DNA (mtDNA) lineage is found to date among several thousands of Europeans and in seven early modern Europeans, interbreeding rates as high as 25% could not be excluded between the two subspecies. In this study, we introduce a realistic model of the range expansion of early modern humans into Europe, and of their competition and potential admixture with local Neanderthals. Under this scenario, which explicitly models the dynamics of Neanderthals' replacement, we estimate that maximum interbreeding rates between the two populations should have been smaller than 0.1%. We indeed show that the absence of Neanderthal mtDNA sequences in Europe is compatible with at most 120 admixture events between the two populations despite a likely cohabitation time of more than 12,000 y. This extremely low number strongly suggests an almost complete sterility between Neanderthal females and modern human males, implying that the two populations were probably distinct biological species. A model of human expansion into Europe reveals almost complete sterility between Neanderthal females and modern human males, implying that the two populations were probably distinct biological species Homo sapiens neanderthalensis (HN) constitute a group of hominids, whose particular morphology developed in Europe during the last 350,000 y under the effect of selection and genetic drift, reaching its final form approximately 130,000 y ago (Homo sapiens sapiens (HS), approximately 45,000 y ago  ConThe additional realism of this model makes it also more complex, and the range expansion and admixture processes will depend on several parameters, like the carrying capacities of the local populations, their intrinsic growth rate, the amount of gene flow between adjacent demes, the local rate of admixture between populations, or the geographical origin of the range expansion. Since it is difficult to explore this complex parameter space, we used archeological and paleodemographic information to calibrate the values of these parameters. For instance, the estimated duration of the replacement process  and 0.09% (scenario E)  . Our simnario E) . This isnario E) C. If thinario E) .Additional complexities of the simulation model could have been envisioned, like the possibility for long-range dispersal, some heterogeneity of the environment leading to different carrying capacities and preferential colonization routes, or uneven migration rates. However, these extra parameters would have been very difficult to calibrate due to the scarcity of paleodemographic data. Moreover, it is likely that they would not have lead to qualitatively different results. For instance, since long-range dispersal speeds up the colonization process , short rCompared to previous models assuming an instantaneous mixing of HN and HS populations  see FigA, we finUnder our model, the progressive range expansion C and theAnother important result of this study is to show that an expanding population or species is likely to have its own genome invaded by that of the invaded population if interbreeding is possible and gradual, which could explain some documented cases of mtDNA introgression e.g., . Our resHomo by modern humans and against a multiregional evolution of H. sapiens  and one potentially occupied by Neanderthals (HN). Given the estimated range distribution of Neanderthals , in agreement with density estimates for Pleistocene hunter-gatherers  used by some previous simulation of modern humans ,, which is of the same order of magnitude as the rare available estimates . Scenario B is identical to scenario A, except that the HS origin is located in Iran. Scenario C uses the same parameters as scenario A, but the HS source is more diffuse and corresponds to a subdivided population of 25 demes  surrounding the source deme defined in scenario A. Scenario D is identical to A, except that the initial HS population is even much more numerous  and occupies all the south area of the HN occupation zone. Scenario E is identical to A, but HSr is here equal to 0.8, which is the maximum growth rate estimated for the Paleolithic human population . For instance, a value of 1/10 means that one admixture event occurred on average in one deme out of ten during the whole cohabitation period between HN and HS.While the γ values are the true parameters of our model, they may not be very telling per se, and we have therefore chosen to quantify levels of interbreeding between populations using another parameterization, which is the average number of admixture events per deme between modern humans and Neanderthals. By performing a large series of simulations, we could find the values of ij,γ the demography of the more than 14,000 demes is thus simulated for 1,600 generations. The density of all demes, the number of migrants exchanged between demes from the same population, and the number of admixture events resulting in gene movements between Neanderthals and modern humans are recorded in a database. This demographic database is then used to simulate the genealogy of samples of 40 genes drawn from 100 demes, representing a total of 4,000 modern human genes distributed over all Europe and corresponding approximately to the current sampling effort of European mtDNA sequence  listed in (357 KB TIF).Click here for additional data file.Figure S2(322 KB TIF).Click here for additional data file.


In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1) although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution.Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1.Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HS1 in apoptosis and for cortactin in cell migration. EMS1 , CTTN, cttn, Amplaxin, see Genecard [EMS1 gene, which is located on chromosome 11q13 [Cortactin . The cein vitro ,23 and tin vitro . Cortactin vitro ). Tyrosiin vitro . Since cin vitro  and as aHS1 . Human Genecard ) was oriGenecard . HS1 oveGenecard , whereasGenecard . Only inGenecard ,30. SecoGenecard , T-cellsGenecard , mast ceGenecard  and erytGenecard , but at Genecard ,23. ThirGenecard , whereasGenecard ,37. FourGenecard  and WEH1Genecard . An HS1 Genecard . FinallyGenecard  and HS1-Genecard ) than thin vitro , although less efficient than cortactin [rd repeat of the F-actin binding domain in a systemic lupus erythematosus (SLE) patient resulting in increased apoptosis after B-cell receptor (BCR) stimulation [Cortactin and HS1 share also remarkable similarities. First, HS1 binds with its DDW-motif directly to Arp2/3 and is involved in Arp2/3 mediated actin polymerization ortactin . Second,ortactin , howevermulation . Fourth,mulation ,45 and tmulation , althougmulation . Finallymulation  and marcmulation , but alsmulation  and carcmulation .Although cortactin and HS1 share a high amino acid sequence and structural similarity, their functions differ considerable. In this paper, we compare their genomic organization in order to provide more insight into their evolution, which may form the basis towards understanding specific functions of both genes. We describe the genomic organization and the exon-intron boundaries for human cortactin. Both the genomic cDNA and deduced amino acid sequences of human cortactin were compared to cortactin and HS1 genes from other species. Genomic comparisons revealed the evolution and underscore the significance of the conserved F-actin binding repeat domain for HS1 and cortactin and the importance of alternative splicing for cortactin function.EMS1 cDNA [EMS1 cDNA sequence revealed homology with two human genomic clones (DDBJ/EMBL/GenBank Accession No. AP000487 and AP000405) . The splice donor and acceptor sequences, the sizes of the introns and exons of the human EMS1/ cortactin gene are provided in the supplementary materials [see We have previously described the isolation and sequencing of the MS1 cDNA ,49 . The seDrosophila melanogaster ) [Xenopus laevis ) [Other cortactin homologues have been reported in mouse , rat 1919, chickgaster ) , and frolaevis ) . We searDrosophila and mosquito) differ considerably with mammalian cortactin, despite the proteins sequences are very similar. Drosophila and mosquito carry 4 repeats in the actin-binding domain. In both species, repeat 1-to-3 and 4 are on separate exons with in mosquito the 4th repeat of the actin binding domain to be encoded by a single 111 bp large exon 2  are available and therefore we were able to reveal their genomic organization using BLASTn. The exon/intron-boundaries were determined and compared to human cortactin see . As sche2 Figure . Both, sBoth nucleotide and amino acid sequence comparisons with cortactin revealed the highest similarity with the hematopoietic lineage cell-specific protein 1 (HS1). So far, HS1 homologues have been reported in human , mouse , rat andX. laevis HS1), the cDNA (and corresponding protein) sequences were deduced from the genomic DNA with BLASTn or were predicted by Ensemble program. In these lower species, two cortactin related proteins exist. To distinguish between cortactin and HS1 variants, only the most conserved N-terminal part of cortactin and HS1 protein variants, including repeat 3 (corresponding to amino acid 1–190 of human cortactin) was used in BLASTp analysis. In each species, one protein variant turned out to be more homologous to human cortactin, and was called cortactin, whereas the other protein variant appeared to be more related to HS1 and was called HS1. This analysis unveiled HS1 proteins with more than 3 repeats in chicken and pufferfish Tetraodon nigroviridis (containing 4 1/2 repeats), pufferfish Takifugi rubripes and Xenopus laevis HS1 (5 1/2 repeats) and zebrafish HS1 (6 1/2 repeats)  genomic sequences in chicken, pufferfish, zebrafish and frog Table  that werMoreover, alignments of the exon/intron boundaries of these HS1 genes to the mammalian HS1 genes see  revealedDrosophila and mosquito cortactin shares putative transcription factors found both in mammalian cortactin and HS1. Thus at least in mammalians, the nature of the promoters seemed to determine the broad distribution of cortactin expression in various tissues except most hematopoietic cells and the limited expression of HS1 to hematopoetic cells.Cortactin is widely expressed in most cell types suggesting to be important for vital functions, while HS1 expression is restricted to hematopoietic cells suggesting to be tailored later in evolution to serve a specific function in these cells. In concordance with their tissue-specific expression pattern, we suppose that their expression might be differently regulated. Therefore, we compared the upstream promoter regions of several cortactin and HS1 genes Figure . The mamth repeat and SV2 lacking the 5th and 6th repeat resulting in a different F-actin binding properties and decreased cell migration [Xenopus laevis ), the SV1-cortactin variant exists despite the splice donor of intron 11 begins with a GT [We recently reported the identification of two alternative splice variants of human cortactin; SV1-cortactin lacking the 6igration . As showigration  except figration . As has igration . Howeverith a GT . Thus, cith a GT . The relith a GT  suggesterd repeat (exon 7) has been found in an SLE patient [rd repeat and its NLS links HS1 to a role in apoptosis, while such a role has not been described for cortactin lacking a NLS. Since the cytoskeleton architecture in hematopoietic lineage cells is very different from that in adherent cells, it is likely that HS1 plays an important role in the construction of tissue-type specific actin networks. Other types of actin cytoskeleton factors, such as the Arp2/3 complex activators of the WASP family have been reported to have distinct tissue specific expression profiles as well. Thus, the apparent role of HS1 in apoptosis is likely due to its actin remodeling related function. Additionally, our genomic comparisons revealed that the 3rd repeat of HS1 corresponds with the 5th repeat of cortactin, and therefore it might be of interest to investigate whether cortactin SV2 variant (lacking the 5th and 6th repeat) might be involved in apoptosis.Alternative splicing also occurs in human HS1. Recently a splice variant lacking the 3 patient , resulti patient . With reth repeat of cortactin has been suggested to be required for F-actin-binding [th repeat. Nonetheless, HS1 does bind to F-actin and activate the Arp2/3 complex, although at a lower efficiency than cortactin [2 binding site in each of its 3 repeats, whereas cortactin has only one in the 4th repeat. PIP2 reduces F-actin cross-linking by cortactin, probably due to competition for the same binding site. Due to its higher affinity for PIP2 [2. This might be of importance in platelets and megakaryocytes where both, cortactin and HS1 are expressed. Taken together, the composition of the repeat domain is also involved in diverting the functions of both genes.The 4-binding . Genomicortactin . This suortactin ,18. In afor PIP2 , HS1 resDrosophila cortactin gene were viable and fertile, except impaired border cell migration during oogenesis [An elegant way to study the function of a protein is to perform loss-of-function experiments. So far, cortactin knock-out models have not yet been generated successfully, because deletion of one allele of cortactin leads to premature differentiation of embryonic stem cells . Howeveogenesis . Down-reogenesis  and in Eogenesis . Mice laogenesis , howeverDrosophila (Dm)), urochordate (sea urchin, (Sp)) and sponge (Sd) cortactin. In this last cluster all the species with only one gene (with the highest similarity with cortactin) are present. This suggests that with the appearance of the vertebrates, an ancestral gene became duplicated to create two genes, which later evolved into cortactin and HS1. This hypothesis is supported by the fact that many genes duplicated at this stage in the evolution, the overall amino-acid sequence in both genes is very similar and the introns are located at the same amino acid position. Furthermore, gene duplication often correlates with a tissue specific expression pattern of the duplicated genes, which is true for mammalian cortactin and HS1.To examine the genesis of the cortactin family, we studied the relationship between the cortactin and HS1 homologues by generating a phylogenetic tree based on a multi-sequence alignment with the ClustalW 1,83 program see . We compFigure Takifugu rubripes contains two cortactin-related genomic sequences both including 5 1/2 repeats. Most likely, an ancestor vertebrate cortactin gene underwent gene duplication. From this moment on during evolution, two cortactin/HS1-releated genes are present in all higher species. One gene evolved to mammalian HS1 with a specific function in apoptosis in hematopoietic cells. For its function, exon 8 and 9 (encoding repeat 3 and 4) were not useful and lost during evolution. However, the HS1 protein in pufferfish Takifugu rubripes and frog Xenopus laevis contains 5 1/2 repeats, while chicken and pufferfish Tetraodon nigroviridis HS1 carries 4 1/2 repeats. It might be of interest to investigate the function of these HS1 proteins and their functional differences to mammalian HS1. The other gene evolved to a ubiquitously expressed mammalian cortactin protein with a vital function in the organization of the cytoskeleton and cell migration. The 6th repeat of cortactin most likely originated from a duplication event of the 5th repeat, since the 6th repeat is most similar to the 5th repeat in all species with 6 1/2 repeats. We recently demonstrated that 6 1/2 repeats are necessary for optimal F-actin cross-linking activity and cell migration, while the splice variant lacking both the 5th and 6th repeats (SV2) was less efficient [th repeat (dotted arrows), since both zebrafish cortactin and HS1 contain 6 1/2 repeats.The genome of pufferfish fficient . Thus, tWe report the genomic organization of cortactin and HS1 genes of several species. These genes display a conserved genomic organization as the coding regions have almost identical exon/intron structure. Comparison of 5' sequences allows possible regulatory elements that stress their specific tissue distribution. Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides insight into the evolution of the conserved actin-binding repeat domain, which forms the basis towards understanding specific functions of both genes. Most likely, both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HS1 in apoptosis and for cortactin in cell migration.To determine the genomic structure of the human cortactin gene, an algorithm was applied based on the consensus sequence of exon-intron junctions ('gt ... ag' rule of intronic sequence) as well as on the codon usage within ORF. Nucleotide sequence comparisons with human cortactin sequences (NCBI, GenBank accession no. M98343) using BLASTn  revealedTo confirm the predicted genomic structure, we determined the intron/exon boundaries using a cloning procedure as described . GenomicBecause no overlapping genomic sequences immediately 5' of the first exon were present in the database, we performed sequence analysis of a 2.7-kb HincII-HincII fragment representing the first exon and its 5'-flanking sequences from cosmid COS-7.12 cloned into pUC18 (p5'EMS_3135). In addition, we sequenced a 5-kb PCR product using a 5'-primer in the vector (within the TET gene) and 3'-primer (p3135p601: 5'-ccgggtcggccctggattcc-3') within exon 1, subcloned in pUC18 (p5'EMS_4911). Nucleotide sequences of both products were compared with the genomic clones representing the cortactin gene present in the NCBI database (Accession number AP000487 (GI 8118774 and GI 6277297) and AP000405 (GI 8118742)) and used to define the 7.4 kb 5'-flanking region. The PROSCAN program  from BIMThe (deduced) protein and genomic sequences of all cortactin and HS1 genes were retrieved from various WEB-sites and their available sequence data are summarized in Table To determine the exon/intron boundaries of all cortactin and HS1 genes, available genomic sequences were subjected to sequence alignments of each species-specific cDNA sequence using the BLAST program of NCBI. Using the same algorithms, as described for human cortactin, the exon/intron-boundaries could be predicted. The complete genomic sequences of the 5' flanking region of cortactin of human, chimpanzee, mouse, rat, fruit fly, and mosquito were determined using the various accession numbers of genomic DNA in Table Sequence alignments were carried out using the BLAST program of NCBI. The multiple sequence alignments of various cortactin proteins were constructed using Basic GeneBee ClustalW 1.83 . The genaa, amino acid(s); bp, base pair(s); BCR, B-cell receptor; EST, expressed sequence tag; HS1, hematopoietic lineage cell-specific protein 1; NLS, nuclear localization signal; RT-PCR, reverse transcriptase polymerase chain reaction; SH3, Src homology; UTR, untranslated region.AGSHvR designed the study on comparative genome analysis, performed database searches, sequence alignments and gene structure prediction and drafted the manuscript. ESS designed, conducted and analyzed the cloning and sequencing of the promoter of human cortactin. VvBvS conducted and analyzed the PCR and sequencing experiments of the exon-intron boundaries of human cortactin and its splice variants. PMK read the manuscript and provided comments. ES helped with writing the paper, provided overall technical guidance and coordination. All authors read and approved the final manuscript.Splice donor and acceptor sequences of cortactin in different species.Click here for fileSplice donor and acceptor sequences of HS1 in different species.Click here for fileMultiple amino acid sequence alignment of cortactin and HS1 homologues.Click here for file


If you could peer inside someone else's head, you'd see a scrunched-up gelatinous mass of tissue, weighing roughly a kilogram, homogeneous to the naked eye—in other words, a brain. The seeming uniformity of the overlying cerebral cortex, which has so outstripped other parts of the brain over the course of evolution that it makes up more than 80% of the brain, is belied by centuries of painstaking neuroscience. Some of the most compelling early evidence that parts of the cortex are specialized in their duties came from gun-shot wounds during the first world war. For instance, bullets lodged in the back of the brain disrupted sight in discrete portions of the visual scene, prompting insights into the localization and function of visual cortex.The study of the front of the brain has a similar history of injury leading to insight. Phineas Gage, a railroad worker, had a 3.5-foot-long tamping iron blow straight through his frontal lobes and turned from a responsible, mild-mannered geek into an unruly exhibitionist overnight. Parts of the prefrontal cortex that he damaged have since been much studied for their involvement in motivation and emotional control.More recent work has implicated other parts of the prefrontal cortex in working memory. Working memory is famously illustrated by your ability to temporarily remember a seven-digit telephone number, roughly the amount of information that you can store on-line in working memory for the duration of a task like phoning for a pizza.Monkeys can be trained to remember information much like you remember a phone number, and then use the memory for gaining a reward . They can learn to remember the specific location of a briefly flashed target on a screen and then, when cued, make an eye movement to look directly at that location. Previous research has shown that neurons in the prefrontal cortex maintain high rates of activity while monkeys remember the target location, and gradually the idea that the prefrontal cortex specializes in maintaining these transient memories has risen to dominance over other ideas about its functions.PLoS Biology, Mikhail Lebedev and his colleagues challenge this prevailing view with evidence that most prefrontal cortex neurons may not be so closely tied to working memory after all. As in previous research, they also trained monkeys to make an eye movement to a remembered target, but instead of only seeing one target, the monkeys saw two potential target locations during the course of the task. The monkeys had to pay attention to one of the potential targets, but this was not necessarily the one they would have responded to and was not the one they had to remember. To perform the task successfully, the animals had to engage their working memory, but most of the neurons the researchers recorded increased their activity selectively to the target that was the focus of attention.In this issue of Despite decades of research, the degree to which one region of the brain can be thought of as dedicated exclusively to a particular function is still much debated. These results do not refute the idea that the prefrontal cortex plays an important role in working memory. However, the authors suggest that this area may be more important in focusing the attention needed to remember that phone number, rather than actually holding that number in your mind.


Close to a quarter of the world's mammals are at high risk of extinction. Save for the periodic “great extinctions,” mammalian extinction has been a relatively rare event in geological terms, with one species disappearing from the fossil record every 1,000 years or so. Over the past 400 years, species have been disappearing 50 times faster than this “background” rate, with one vanishing every sixteen years. Human population growth and all its consequences—habitat destruction, propagation of invasive species, poaching—are largely to blame. Top predators often suffer heavily from encounters with humans, especially when those predators are perceived as economic threats. Thirty-four Mexican gray wolves have been reintroduced in Arizona since 1998, and five have been shot, reportedly by ranchers.Species in the most densely populated areas are expected to face the greatest risk, yet some survive while others perish, suggesting biological factors play a role in their fate. If, for example, the same external force drastically reduces populations of species with different biological profiles, then a species with a relatively short gestation period may stand a better chance of recovering than a long-gestating species.Effective conservation strategies depend on understanding which factors are likely to increase extinction risk, but it's unclear how important intrinsic biological traits are relative to external pressures from humans and whether biology's influence on survival depends on the intensity of the threat. Ecologists often use human population density as a proxy for anthropogenic threats such as habitat destruction and hunting. To tease out the relative importance of all these factors, Marcel Cardillo et al. analyzed the impact of various biological traits and human population density on extinction risk in the mammal order Carnivora, which includes the red panda, lion, and members of the photogenic weasel-like viverrid family. By identifying the most salient factors in predicting extinction, the authors have created a model to identify those species at greatest risk.The biology of a species combined with human population density, the researchers found, is a stronger predictor of risk than exposure to humans alone; those biological traits that increase risk vary depending on a species' exposure to human populations. Carnivores with low exposure to humans, for example, are likely to be at greater risk if their population density is low and they have small ranges, possibly because this makes them more vulnerable to loss of habitat. Species living near densely populated human areas must often contend with hunting and other direct threats on top of habitat loss and are more at risk if they also have long gestation periods—they can't repopulate fast enough to offset the additional pressures. Based on projected human population growth, this model predicts the addition of a number of species—mostly from Africa, where population growth rates largely exceed the global average—to the endangered list by the year 2030. Most of these species—including African viverrids such as the common genet, which not only lives in areas where human populations are rapidly expanding but is also biologically predisposed to decline—are currently considered a low conservation priority.While it's possible that the direct effects of human population density are past—that is, species most sensitive to human incursions are already gone—human population density likely modulates biology. That might explain why gestation length didn't predict risk for species living in sparsely populated areas—all else being equal, their numbers remained relatively stable. A species with a small population forebodes a high extinction risk regardless of human density, though species with long gestation periods, again, face greater danger in the company of humans.before a species reaches the brink of extinction. “There is no room for complacency about the security of species,” the authors warn, “simply because they are not currently considered threatened.”Altogether, these results suggest that as human population pressures increase, the importance of species-specific biology in predicting extinction risk also increases, with biology affecting which species are most vulnerable to external threats. With most conservation efforts focused on damage control, these findings make the case for interceding