In this review, we discuss estrogen actions on mitochondrial function and the possible implications on cell growth. Mitochondria are important targets of estrogen action. Therefore, an in-depth analysis of interaction between estrogen and mitochondria; and mitochondrial signaling to nucleus are pertinent to the development of new therapy strategies for the treatment of estrogen-dependent diseases related to mitochondrial disorders, including cancer. In contrast, "non-genomic" effects of estrogen are characterized by a rapid onset of action within seconds to minutes after hormone exposure through post-translation modification of signaling proteins. ER-mediated signaling pathways are considered to support the growth of normal, preneoplastic and neoplastic cells m, accumulation is reported to promote the generation of ROS [2+]m was reported to stimulate H2O2 production by rat brain mitochondria in the presence of rotenone [2+]m with E2 (100 pg/ml) treatment of MCF7 cells [2+]m with E2 (10 ng/ml) treatment of hippocampal neurons [2+]m is not clear, however, the inhibition of Na-dependent Ca2+ efflux from mitochondria was reported to increase calcium retention in E2 (1 nM) treated synaptosomal mitochondria m, it is possible for estrogen to signal the formation of mitochondrial ROS through a similar mechanism.Mitochondrial Can of ROS . For exarotenone . Using cF7 cells . Another neurons . The mecchondria . There irylation . A study and ROS . Interes and ROS . Since em and/or [Ca2+]m in favor of ROS generation.The inhibition of respiratory complex I is known to favor ROS generation. Rat brain mitochondria that respired on complex I substrates produced a substantial level of ROS when inhibited with rotenone concentrations as low as 20 nM . Since e2•- which is dismutated into the membrane permeable second messenger H2O2 m could stimulate mtNOS activity ultimately leading to the generation of ROS via NO-dependent inhibition of cytochrome c oxidase [Several isoforms of the enzyme nitric oxide synthase (NOS) are reported to exist which include inducible-(i), endothelial-(e), neuronal-(n), and mitochondrial-(mt) NOS. Estrogen has been reported to induce various isoforms of NOS. The activity of eNOS is modulated by estrogen in human aortic endothelial cells, uterine artery, heart, and skeletal muscle ,119. Estger H2O2 . Since eLa cells . We have oxidase .2O2 and/or O2•- has been demonstrated to stimulate cell growth in a variety of cell types including muscle cells, fibroblasts, amnion cells, prostate cancer cells, and aortic endothelial cells [i) mitochondria are the principal source of intracellular ROS in epithelial cells. (ii) the growth of adenocarcinomas occur in tissue of epithelial cell origin.The rapid stimulation of intracellular ROS by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and nerve growth factor (NGF) suggests that this underlying mechanism of cell growth may be shared with other growth factors including estrogen . Exogenoal cells . The molal cells . The proal cells . Althoug2O2 and NO have been demonstrated to stimulate mitochondrial biogenesis, a process that depends on the flow of molecules into and out of the organelle [2O2 levels. In addition, MnSOD displayed an increased pattern of activity similar to mtNOS [2•- production and therefore MnSOD may dismutate O2•- generated by NO-dependent inhibition into the signaling molecule H2O2. Ethinyl estradiol, E2, and estrogen catechol metabolites at a dose of 0.25 to 5 μM are reported to increase mitochondrial O2•- in cultured rat hepatocytes and HepG2 cells [2•- is not known at this time, ROS has been demonstrated to modulate ER protein expression in various cell lines. Treatment of human breast cancer cells MCF7 and T-47D with H2O2 (2.5 μM) increased the protein level of ERβ [A characteristic of rapidly dividing cancer cells is their capacity to produce significant amounts of intracellular ROS, which has been implicated in the promotion of accelerated cell cycle activity in neoplastic cells. Mitochondria have long been suspected to play a role in the development and progression of cancers. The ROS molecules Hrganelle ,129. Sinto mtNOS . NO has G2 cells . Althougl of ERβ . In addiin vitro estrogen-induced proliferation of Syrian hamster renal proximal tubular cells [2•- from human fibroblast cells. Co-treatment with an inhibitor of IL-1β and TNF-α synthesis, pentoxifylline, inhibited stilbene estrogen-induced increase in myeloperoxidase activities, 8-hydroxydeoxyguanosine (8-OHdG) formation, mutations in the testicular genome, and prevented estrogen-induced testicular preneoplastic lesions [Evidence for the involvement of redox signaling with estrogen-induced cell proliferation has been demonstrated in several studies. Liposomes containing SOD or catalase inhibited ar cells . The cyt lesions . Recentl lesions ,135. We  lesions . Therefo2•- production [Oxidative stress has been shown to affect mitochondrial proteins of chronically estrogenized Syrian hamster kidney. A decrease in thiol/sulfhydryl groups was reported in the mitochondrial fraction at a preneoplastic stage of carcinogenesis . Estrogeoduction . Evidencoduction . Since ooduction . Since Moduction , the abs1 to S phase transition, cell growth, and transcription of cell cycle genes was induced by TrxR2N expression. TrxR2DN expression was suggested to increase intracellular H2O2, which in turn signaled cell proliferation. Although it is not clear whether estrogen can modulate the H2O2 levels of mitochondria, estrogen (10 nM–100 nM) treatment of primary human endometrial stromal cells in vitro show an increase in Trx protein and mRNA which implicate Trx involvement in cell growth and differentiation of estrogen responsive tissue [The mitochondrial thioredoxin system has been demonstrated to play a role in cell cycle progression. In general, the two antioxidant oxidoreductase enzymes thioredoxin (Trx) and thioredoxin reductase (TrxR) that compose the system modulate signal transduction properties of ROS by the reduction of intracellular disulfides. Trx acts as a protein disulfide reductant for ribonucleotide reductase and several transcription factors including p53, NF-κB, and AP-1 . Once oxe tissue . Alterate tissue . Whethernd messenger since they are short-lived (rapidly generated and degraded), produced in response to a stimulus, highly diffusible (H2O2), and ubiquitously present in most cell types. It is not known whether mitochondrial ROS like H2O2 are involved in signaling pathways that control estrogen-induced cell proliferation. In this section, we provide evidence in support of redox signaling pathways of mitochondrial origin, which may be involved in cell cycle progression of estrogen-dependent cells.Mitochondrial ROS fulfill the characteristics of a 2Protein kinases are known to participate in phosphorylation signal cascades, however, zinc finger domains contained in some proteins may allow them to participate in redox signaling networks. Zinc finger structures within a protein consist of at least two zinc-coordinated thiolates. Upon oxidation zinc is released from the protein, which converts cysteine thiol groups to disulfide. A conformational change in the protein may result in either its activation or inhibition. There are several protein kinases such as a-raf and PKC that contain zinc finger domains. In addition, zinc finger domains are also found in hormone receptors such as the GR and ER . Both PK1 restriction point [1 checkpoint are independent from the MAPK signaling pathway, a new question rises of whether mitochondria can modulate cell growth by interacting with the cytoskeleton. Mitochondria are reported to be associated with three major cytoskeletal structures, which include microtubules, microfilaments of actin, and intermediate filaments [m by its interactions with VDAC [Mechanical signals associated with cytoskeletal tension generation and cytoskeleton restructuring are a requirement for anchorage dependent cells to pass through the late Gon point . Since tilaments . Mitochoilaments . The assith VDAC .Epithelial cell spreading results from the binding of integrins to the extracellular matrix which depends on the actin cytoskeleton ,163. The1 phase of the cell cycle but also starts in the late S phase [Mitochondria biogenesis is reported to occur in the G S phase . Althoug S phase . This ch S phase . Estroge S phase . Thus, e1, and core protein I of the cytochrome bc1 complex; the α and β subunits of F1 ATPase and subunit IV of cytorochrome oxidase [The mitochondrial protein cytochrome c is known to be released to the cytosol where it initiates a signal for apoptosis. Given the role of cytochrome c the existence of other mitochondrial protein signaling molecules is a likely possibility that could mediate a diverse number of cellular processes including cell growth. It has been shown that mitochondria have the capability to export mitochondrial-matrix proteins to other cellular compartments such as the nucleus, peroxisome, endoplasmic reticulum, and secretory vesicles . For exa oxidase . The mol oxidase . It was  oxidase ,178. Sin oxidase . In addi oxidase . Based o2+ dependent neutral protease [Several proteases are known to exist in the mitochondrial matrix such as ATP-dependent human Lon protease, ATP-dependent human Clp proteinase chain P (hClpP), and Caprotease -183. Sinprotease . Low molprotease . The 44-protease . It may protease . A signiprotease . Thus, tIn summary, mitochondria are a major target of estrogen. It appears that mitochondria through its interaction with the cytoskeleton, export of cleaved signaling peptides, and/or generation of ROS may transduce signals to the nucleus for the activation of transcription factors, such as, AP-1, NF-κB, and CREB involved in the cell cycle progression of estrogen-dependent cells. These interactions between estrogen and mitochondria merit furture investigations, which may shed new light on the role of mitochondria in cell growth.


The visit to the emergency department (ED) constitutes a brief, yet an important point in the continuum of medical care. The aim of our study was to evaluate the continuity of care of adult ED visitors.We retrospectively reviewed all ED discharge summaries for over a month 's period. The ED chart, referral letter and the patient's primary care file were reviewed. Data collected included: age, gender, date and hour of ED visit, documentation of ED referral and ED discharge letter in the primary care file.359 visits were eligible for the study. 192 (53.5%) of the patients were women, average age 54.1 ± 18.7 years (mean ± SD). 214 (59.6%) of the visits were during working hours of primary care clinics ("working hours"), while the rest were "out of hours" visits. Only 196 (54.6%) of patients had a referral letter, usually from their family physician. A third (71/214) of "working hours" visits were self referrals, the rate rose to 63.5% (92/145) of "out of hours" visits (p < 0.0001). The ED discharge letter was found in 50% (179/359) of the primary care files. A follow-up visit was documented in only 31% (111/359). Neither follow up visit nor discharge letter were found in 43% of the files (153/359).We have found a high rate of ED self referrals throughout the day together with low documentation rates of ED visits in the primary care charts. Our findings point to a poor continuity of care of ED attendees. The emergency department (ED) is intended to treat medical urgencies or emergencies, but a large proportion of visits are due to problems that could be treated in the primary care setting ,2. ED seThe visit to the ED constitutes a brief, yet an important point in the continuum of medical care. In today's era of cost effectiveness and increasingly competent primary care physicians, ambulatory investigation, treatment and follow-up have largely replaced prolonged and costly hospitalizations ,4. The EThe continuation of treatment between hospital departments and the primary care physician had been issued in several studies using discharge letters audit -7. RavalTo evaluate the continuity of care after ED visits, we evaluated the ED referral and discharge letters, their content, and the documentation of the ED visit in the patients' primary care files. We have focused on discharges from the internal medicine ED. We expected that in these cases the patients would be followed up by their family practitioner.The study was conducted in the district medical center (Kaplan), serving more than 500,000 inhabitants, and in 12 primary care clinics (32 family practitioners), of The Clalit Health Services in the Rehovot region, Israel.In Israel the entire population have a national health insurance by law and each citizen can choose to be a member of one of four HMOs. Every member of the Clalit Health Services, the largest HMO in Israel, is registered to a single family physician, and have a medical record in his physician's clinic. Visits to the emergency department are regulated in the national health insurance law. A referral by a physician or by ambulance is free of charge, but this referral should be with a referral letter and not by a phone call to the ED or to the patient. A self referral may cost the patient a co-payment of up to 100 USD.We reviewed retrospectively all the charts of the ED visits for a period of one month, excluding the visits to the pediatric and the gynecologic-obstetrics EDs (see flow-chart 1). 5,898 visits documented that month, resulted in 4,256 discharges and 1,642 hospitalizations. There were 1,564 discharges from the general ED. Trauma, surgery and orthopedics accounted 2,209 discharges and the rest 483 were from other specialties .Inclusion criteria were: visit to the general ED, age above 18 years, discharge to the community  at that visit, living and getting medical care in a family medicine group practice in the Rehovot region. Visits due to accidents, trauma, surgery, orthopedics, ENT, ophthalmology and other specialities were excluded from the study. The 1,564 discharges from the general ED were reviewed and 359 were found to be eligible to this study.Two physicians reviewed independently each ED medical chart. Data extracted included: age and gender of the patient, attendance date and hour, self referral, or a referral by a physician and the final diagnosis in the discharge letter. In cases of referrals the content and format of the referral letter were assessed, including: hand writing quality and whether the referring physician referred the patient with a specific question .Continuity of communication and care: The primary care files of ED visitors were retrieved and checked for the existence of the ED discharge letter and comments about the visit in the follow-up chart. If the discharge letter and / or any comment on the ED visit in the follow-up chart had been found the case was defined as "a case with good continuity of care".The cases in which the family physician was the referring physician we looked for documentation of the encounter prior to ED attendance."working hours" visits – when the visit took place during working hours of primary care clinics in the community , and "out of hours" visits when primary care clinics in the community are closed .Visits to the ED were divided into A referral letter was defined as "any document written by a medical authority in the community prior to the index ED visit", including referrals from family practitioners, other practitioners in the community and arrival by an ambulance.A recurrent visit to the ED was defined as a patient's visit to ED within less than two weeks from a previous visit with the same complaint, when in both cases the patient was not hospitalized.Diagnoses at discharge were coded for a specific diagnosis and for the system involved according to the ICPC coding system.Statistical analysis: Data was analyzed using distribution analysis and χ2 tests to investigate the association between categorized variables. Student's t tests were used to analyze continuous variables. The analysis was performed using the SPSS package.During the study period there were 359 ED visits that were eligible to be included in the study  patients had a referral letter, the rest were self-referrals. Referral letters were mainly from the family practitioner , 14 (7%) from other practitioners in the community, and 35 (18%) of referrals were by ambulance. The referral letters from the community were legible in 43.4% (70/161), 46.5% (75/161) were barely legible and 10% (16/161) illegible. In only 25/161 letters  a specific question was asked by the referring physician and in another 32 (20.2%) there was only a general question.The main diagnostic groups according to the ICPC were: respiratory (15.7%), digestive system (18.1%), musculo-skeletal (15.2%) and cardio-vascular (11%). In 9.6% of the cases the discharge letter did not contain a specific diagnosis and the diagnosis fell in the "general" category. The most common specific diagnoses were: chest pain (5.9%), abdominal pain (3.9%), other respiratory tract infections (3.7%), asthma (3.1%), back pain (2.8%), COPD exacerbation (2.8%) headache (2.5%), nephrolithiasis (2.5%), vertigo or dizziness (2.5%) and gastroenteritis (2.3%).The "out of hours" visitors tended to be younger   of primary care files. The ED discharge letter was found in 50% (179/359) of the primary care files. A follow-up visit was documented in only 31% (111/359). Neither follow up visits nor discharge letters were found in 43% of the files (153/359). No associations between clinic characteristics  or family practitioner qualification and ED visit documentation was found.The Emergency Department (ED) acts as a link between community and hospital based medicine. In Israel a patient who needs non elective admission to a hospital unit must pass through the ED, either with a referral note from a medical practitioner, or as a self referral. Most ED visits, however, do not result in hospitalization, and many could be regarded as primary health care problems ,2,9. TheThe increasing role played by the ED in treating primary care problems has been discussed in a number of recent articles -11.One aspect, which is important to the ED team, is the logistics and manpower needed to optimize the treatment of these non-urgent patients in ways that will not interfere with emergencies yet providing them adequate care. It is unclear whether the capability and quality of primary care services in the ED should be improved and compete with the community family physicians. This is true especially in Israel where there is a universal national health insurance and every patient can have a personal family practitioner.The continuity of comprehensive management is expected from the family practitioner, and is gaining importance nowadays . To achiThe modes of communication are the referral letter and the discharge letter. We have found that the referral letter can be improved both in style (printed instead of illegible hand writing) and content . These problems exist in discharge letters as well .Documentation in the primary care file was poor, only one third of referrals were documented and less than 60% of discharges. This figure is between the 27%–77% that was found by others [It is well known that medical notes are poor in other areas, Miller et al  found doWith the introduction of computerized medical files in primary care clinics in our region, the need of "double writing" will disappear, and this in turn should dramatically improve documentation rate of referrals and discharges to the ED; particularly if a computerized reminder system is used to encourage follow up of referrals by the family practitioner.Israeli health care system works in regard to ED use quite different from the US and other countries. Likewhise these results may not automatically be generalized to other health care systems. This study described the written communication between the emergency department and the primary care physician, which is the first and mandatory step in establishing continuation of care. This is only one of the four dimensions of continuity of care in family practice: chronological, geographical, interdisciplinary, and interpersonal . Each ofED visits may have important implications for the patient and his family practitioner. The high rate of ED self referrals together with low documentation rates of ED visits in the primary care charts result in poor continuity of care of ED visitors.None declared.All authors read and approved the final manuscript.VS Conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. KE Participated in the statistical analysis, interpretation of data and draft of the manuscript.OY participated in the design of the study, data collection and interpretetio hand draft of the manuscript. SN participated in the design of the study, interpretation of data and draft of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The brain-derived protein S100B has been shown to be a useful marker of brain injury of different etiologies. Cognitive dysfunction after cardiac surgery using cardiopulmonary bypass has been reported to occur in up to 70% of patients. In this study we tried to evaluate S100B as a marker for cognitive dysfunction after coronary bypass surgery with cardiopulmonary bypass in a model where the inflow of S100B from shed mediastinal blood was corrected for.56 patients scheduled for coronary artery bypass grafting underwent prospective neuropsychological testing. The test scores were standardized and an impairment index was constructed. S100B was sampled at the end of surgery, hourly for the first 6 hours, and then 8, 10, 15, 24 and 48 hours after surgery. None of the patients received autotransfusion.In simple linear analysis, no significant relation was found between S100B levels and neuropsychological outcome. In a backwards stepwise regression analysis the three variables, S100B levels at the end of cardiopulmonary bypass, S100B levels 1 hour later and the age of the patients were found to explain part of the neuropsychological deterioration .In this study we found that S100B levels 1 hour after surgery seem to be the most informative. Our attempt to control the increased levels of S100B caused by contamination from the surgical field did not yield different results. We conclude that the clinical value of S100B as a predictive measurement of postoperative cognitive dysfunction after cardiac surgery is limited. Despite the fact that incidence figures between 4–79% have been reported for cognitive dysfunction after cardiac surgery , diagnosSeveral studies in humans suffering from stroke of different ethiologies, have shown a rather strong correlation between serum levels of S100B and size of lesion(s) as well as outcome -17.Lately a number of studies have addressed the question whether S100B can be considered as a marker for cognitive dysfunction after cardiac surgery; however the conclusions presented are disparate. One major reason for this could be the fact that S100B is present in high concentrations in shed mediastinal blood that is retransfused to the patient by cardiotomy suction and autotransfusion, thus obscuring the measured levels of S100B early after surgery. To date, only one study have been published where this contamination was taken into account . We receThe study group comprised 56 patients who underwent coronary artery bypass graft (CABG) surgery at the Division of Cardiac Surgery, University Hospital MAS, Malmoe, Sweden and Department of Cardiothoracic Surgery, Lund University Hospital, Lund, Sweden. Only patients planned for elective CABG with cardiopulmonary bypass (CPB) as their sole procedure were included. The study protocol was approved by the local ethics committee, and patients gave a written informed consent before the study protocol was initiated. Patients with a history of stroke, transient ischemic attack (TIA), reversible neurological disorder (RIND), known carotid artery disease or other brain diseases were excluded. In order to avoid possible influence of renal disorder on the elimination of S100B, patients with known renal failure (Creatinin > 160 μmol/L) were excluded.The patients were examined for signs of neurological dysfunctions daily during the hospital stay by either experienced cardiac anesthesiologists or by experienced cardiac surgeons.®, Roche, Basel, Switzerland) or propofol  10 mg/kg. It was subsequently maintained with fentanyl 10 μg/kg , a continuous infusion of propofol 3–6 mg/kg/h or inhalation of isoflurane 0,5–1% . Nitrous oxide  was used before CPB but not during or after CPB.Anesthesia was induced with midazolam 3–5 mg iv  and administered in the ascending aorta and the anastomosed vein-grafts intermittently.CABG surgery was performed during aortic cross clamping with the distal anastomosis preceding the proximal anastomosis. A tangential occluder replaced the cross-clamp during the proximal anastomosis. Antegrade cold S:t Thomas crystalloid cardioplegia was used (Cardioplegi2 at normothermia. The perfusate was cooled to approximately 32°C. Heparin (400 U/kg bodyweight) was given prior to cannulation and reversed with equal doses of protamine sulphate at decannulation.Perfusion was performed with a roller pump . The perfusion catheters and circuit were made of polyvinylchloride in the line and silicon in the pumphead. The arterial cannulation was made in the ascending aorta and venous cannulation in the right atrium by a two-stage venous cannula. All circuits contained a heparin-coated 40 μm arterial filter  and a membrane oxygenator . The circuit was primed with approximately 1000 ml of Ringer's lactate (Pharmacia-Upjohn), 250 ml 15% Mannitol (Pharmacia-Upjohn) and 75 mmol Addex tromethamine (Pharmacia-Upjohn). Perfusion flow was non-pulsatile with a flow rate of 2.4 l/min/mAfter surgery, the patients patient were transferred to the ICU for recovery and enrolled in the sampling scheme for S100B analysis. None of the patients received autotransfusion.Serum for S100B analysis was sampled before surgery, at the end of CPB, and then 1, 2, 3, 4, 5, 6, 8, 10, 15, 24 and 48 hours after surgery. The S100B levels at these time points will be referred to as T0, T1, T2....T48. Blood samples, both arterial and venous samples, were cooled and centrifuged within 5 hours. All samples were measured by a monoclonal two-site immunoluminometric assay .Since early levels of S100B are contaminated by S100B from cardiotomy suction, an attempt was made to exclude this S100B from the levels measured one and two hours after surgery. Assuming that all of the measured S100B at the termination of CPB was a contamination, this non-cerebral S100B was eliminated with a half-life of 25 minutes, as illustrated in figure e is the estimated true levels of S100B at time t, Ct = the measured concentration of S100B at T1 or T2, C0 the concentration at the end of CPB, t the time after T0 and t1/2 the half-life of S100B.where CWe have earlier suggested that the elimination rate could be used as another measure of cerebral release , and theThe patients underwent neuropsychological testing by the same trained neuropsychologist 1–2 days before and 5–7 days after surgery. The tests used were: Mental Control, Figural Memory, Logical Memory (A/B), Visual Reproduction, Rey Auditory/Verbal Learning Test (RAVLT), Trail Making A, Trail Making B, Digit Symbol, Digit Span, Visual Memory Span, Visual Paired Associates II or Verbal Paired Associates I and RAVLT, Delayed Retention. The tests were chosen from the Wechsler Memory Scale-Revised (WMS-R), the Wechsler Adult Intelligence Scale (WAIS – R) and the Halsted-Reitan Neuropsychological Battery ,21.Differences for each sub-test were first calculated and then standardized to z-values. All sub-tests were then aggregated to create an impairment index . The impAll results were analyzed with the Statistica version 5.0 for PC. Regression analysis was performed with the least square method with a casewise deletion of missing data. For the multiple regression a backwards stepwise regression was performed to determine which variables to include in the final analysis. The initial variables included in this analysis were age, gender, perfusion time, years of education, and S100B levels at all sampling times. A p-value < 0,05 was considered significant. Unless otherwise stated numerical values are presented as mean ± 1 standard deviation.Patient demographics are presented in table The appearance of S100B protein followed the same pattern in all patients with high levels at the end of CPB and a decrease or slight increase the first hour figure . ThereafWe found a correlation between patient age and S100B levels measured up to 24 hours after bypass Table . NeitherIn multiple regression analysis, measured S100B levels at the end of CPB (T0), one hour later (T1) and age were found to explain part of the neuropsychological impairment  declare that they have no competing interests.HJ: Principal investigator recruited, enrolled patients analyzed and wrote the paperPJ: Had an integral role in the planning and the analysis, also help with writingMB: Designed neuropsychological test battery. Analyzed neuropsychological dataCB: Performed all neuropsychological testing with patientsCA: Did the S100B-analyzesSB: Mentor an principal leader of the project, who also helped with the writing.The pre-publication history for this paper can be accessed here:


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  supplemented with 10% fetal bovine serum (FBS)  and penicillin (100 U/ml)/streptomycin (100 μg/ml) . Passaging of cells was performed every 3–4 days with cells grown to 80% confluency in a 10 cm cell culture dish . 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  with each of the five DNA repair genes were grown in LB-broth with ampicillin . 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 . 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  and hNTH  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  and then exposed to high performance autoradiography film . 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  and protein concentrations were determined using the DC protein assay . 20 ug of protein were loaded into individual lanes of a NuPage Bis-Tris Gel . The gel was then transferred to nitrocellulose paper . The membranes were then blocked with 1% blocking solution  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  located in a 37°C incubator. The oxygen chamber was then infused with 95% OThe SRB assay , 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 . 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 . 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 . 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  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  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  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. 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 . 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 : water  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  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  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.