There is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation.Semen samples from 55 men attending the Histology-Embryology Laboratory of Sfax Faculty of Medicine, Tunisia, for semen investigations were analysed for sperm DNA fragmentation and oxidation using flow cytometry. The Sperm was also assessed spectrophotometrically for malondialdehyde formation.Within the studied group, 21 patients were nonasthenozoospermic (sperm motility ≥ 50%) and 34 patients were considered asthenozoospermic (sperm motility < 50%). A positive correlation was found between sperm DNA fragmentation and oxidation . We also found a negative correlation between sperm DNA fragmentation and some sperm parameters: total motility , rapid progressive motility (type a motility) , slow progressive motility (type b motility) , and vitality . Sperm DNA fragmentation was positively correlated with coiled tail . The two parameters that were found to be correlated with oxidative DNA damage were leucocytes concentrations  and broken neck . Sperm MDA levels were negatively correlated with sperm concentration , total motility  and type a motility ; but not correlated with DNA fragmentation and DNA oxidation.Our results support the evidence that oxidative stress plays a key role in inducing DNA damage; but nuclear alterations and malondialdehyde don't seem to be synchronous. Infertility affects around 15% of couples in reproductive age and male factor is a major contributor by approximately half of these cases ,3. DetecLipid peroxidation is one of the deleterious effects of ROS and is considered as an indicator of membrane polyunsaturated fatty acid oxidation -8. MalonMoreover, it was reported that ROS, in addition to reacting with the polyunsaturated fatty acids, might also react with DNA nucleotides leading to base modifications particularly 8-hydroxy-2'-deoxyguanosine (8-oxoguanine) formation and DNA fragmentation -13. The In this study we aimed to evaluate the concentration of sperm MDA and the levels of DNA fragmentation and oxidation in infertile men and to investigate the eventual correlations between routine semen parameters, sperm DNA damage and malondialdehyde formation.This study was approved by the Institutional Review Board of Sfax Faculty of Medicine, Tunisia. A total of 55 men attending the Histology-Embryology Laboratory of Sfax Faculty of Medicine (Tunisia) for semen investigations were included in this study. Written informed consent was obtained from all the subjects for publication of this case report. The patients neither had any urogenital diseases or infections nor did they undergo x- ray or chemotherapy. They were aged between 26 and 62 years old with a mean age (± SD) of 37.49 ± 0.89 years. According to the World Health Organization (WHO) criteria , semen sSemen samples were obtained by masturbation into sterile containers after 3-5 days of sexual abstinence and left to liquefy at 37°C. Basic semen analyses were performed within1 hour of collection and consisted in the measurement of semen volume, sperm concentration (hemocytometer method), motility , slow progressive (type b), non progressive (type c)), vitality and morphology. Semen samples were also assessed for leucocytes concentration using peroxidase method. All parameters were carried out according to the WHO guidelines .Sperm DNA fragmentation and oxidation were evaluated in fresh semen. The remainder of the sample was aliquoted and stored at -80°C for further analysis of malondialdheyde levels.6 cells were washed with phosphate-buffered saline  then fixed with 200 μl of 4% paraformaldehyde for 1 hour at room temperature in the dark. Afterwards, sperm cells were washed with 1 × PBS and permeabilized using 0.1% Triton X-100 in 0.1% sodium citrate for 15 minutes on ice. After washing with PBS, sperm DNA was labelled by incubating spermatozoa with 50 μl of the TUNEL reaction mixture (Tdt enzyme and FITC-labelled nucleotides) in a humidified atmosphere for 60 minutes at 37°C in the dark, with mixing each 15 minutes. Washed and labelled sperm cells were then resuspended in 1xPBS for flow cytometry analysis. A negative control (sample without the addition of Tdt enzyme) and a positive control  for 10 minutes at room temperature to generate DNA strand breaks) were also assessed by TUNEL assay.For the evaluation of DNA fragmentation, a commercial kit  based on an enzymatic reaction of labelling free 3'-OH termini was used. As previously described , 3.106 c6 spermatozoa was washed with 1xPBS, fixed, permeabilized with ice-cold 70% ethanol and kept 1 hour at -20°C. Fixed cells were centrifuged at 1600 rpm for 5 minutes, washed with PBS, then resuspended in 1 ml wash solution  and pelleted at 1600rpm/min for 5 minutes. Fifty μl FITC conjugate were incubated for 1 hour with pelleted sperm cells in the dark at room temperature, with mixing every 15 minutes. Finally, cells were washed, resuspended in 500 μl 1xPBS for flow cytometry analysis. For the positive control, sperm cells were washed in 1ml PBS then incubated in H2O2 (4 M) solution for 1 hour at 37°C. The negative control consisted of selected spermatozoa from healthy men. This control had a percentage of labelled sperm < 2%.We used the oxyDNA kit  which is specific for detection of 8-oxoguanine as one of the major studied oxidised nucleotides. The test is based on the direct binding of a probe conjugated to fluorescein isothiocyanate (FITC-conjugate) to DNA adduct 8-oxoguanine. In brief, as previously described ,18, one Flow cytometric analysis was carried out using an EPICS XL flow cytometer (Beckman Coulter) equipped with a 15mW argon-ion laser for excitation at 488 nm. At least 10000 events per sample were analysed. Light-scattering and fluorescence data were obtained at a flxed gain setting in logarithmic mode. Debris was excluded by establishing a region around the population of interest on the basis of light scatter characteristics (forward-angle light scatter (FSC) vs. side-angle light scatter (SSC). The percentage of labelled sperm was characterized by identifying a region that included > 90% of events in the frequency histogram of the positive controls both in the assessments of DNA fragmentation and oxidation . The mixture was then treated in a boiling water bath for 15 min. After cooling, samples were centrifuged at 4,000 × g for 10 min. The content of MDA was measured spectrophotometrically by the determination of the supernatant absorbance at 532 nm. The MDA fluorescence intensity of spermatozoa was determined using various concentrations of tetraethoxypropane as standards. The results were expressed as nmol MDA/107 cells. The MDA assay used had an intra-assay coefficient of variation of 7.4%.Lipid peroxidation in spermatozoa was measured in 43 semen samples with the commonly used thiobarbituric acid reactive substances (TBARS) method, according to Yagi . It was The SPSS  software (version 18) was used for statistical analyses. Non-parametric test (Mann-Whitney U-test) was performed to compare sperm parameters and flow cytometry data between nonasthenozoospermic and asthenozoospermic semen. The relationship between semen parameters, DNA fragmentation, DNA oxidation and MDA levels was analysed using Spearman's correlation coefficients. We applied linear regression to determine the correlation between leucocytes concentration and sperm damage parameters (DNA fragmentation and oxidation and MDA) with adjustment of sperm motility and concentration. Statistical significance was established for a p-value of < 0.05.The mean values (± SD) and ranges of routine semen parameters, MDA levels and percentage of sperm DNA damages are summarized in Table 7 sperm; p = 0.008 and 28.01 ± 2.23% vs 19.34 ± 2.55%; p = 0.006 respectively). However, the levels of 8-oxoguanine (± SD) were not significantly different when comparing the two groups  respectively.The means (± SD) of sperm MDA concentrations and of DNA fragmentation percentage were significantly higher in the asthenozoospermic group than in nonasthenozoospermic one  Figure .Numerous studies have focused on the relationship between sperm DNA damage and standard semen parameters -24 but sAs regards the relationship between sperm DNA damages and semen quality, we found that DNA fragmentation was higher in asthenozoospermic patients than that in nonasthenzoospermic ones. The high prevalence of DNA fragmentation in dyspermic semen samples as compared to normospermic was reported previously -24,29. SBesides the evaluation of sperm DNA fragmentation and oxidation, we used the malondialdehyde (MDA) assay to measure lipid peroxidation in spermatozoa. This assay was chosen, first for its simplicity and sensitivity ; SecondlAs expected, our results showed that sperm MDA concentration was significantly higher in asthenozoospermic than in nonasthenzoospermic patients. Besides, we found that MDA levels were negatively correlated to sperm concentration and motility which was consistent with previous reports ,40. In oAmong the interesting findings of our study, a significant correlation was observed between sperm DNA fragmentation and oxidative DNA damage. This result was similar to that reported in a recent study by Aitken et al.,  suggestiThe second important result we reported for the first time was the significant correlation between leucocytes concentration and 8-oxoguanine. However, leucocytes concentration was correlated neither to DNA fragmentation nor to MDA levels. This makes us suggest a direct implication of leucocytes, as an exogenous factor, in generating base modifications, and a more susceptibility of DNA bases to ROS action; obviously, 8-oxoguanine formation reflects a direct and specific action of ROS on sperm DNA . Our datFrom literature data, many hypotheses were postulated to explain the origin of sperm DNA damage such as defective chromatin packaging during spermiogenesis ,43,46, aSperm damages could be also caused by different events related to iatrogenic, idiopathic, environmental and pathological factors and to lifestyle ; howeverSperm DNA damage is thought to be detrimental to ART outcomes, particularly to pregnancy rates ,13,31,28Finally, it should be noted that the techniques used to assess sperm DNA integrity need more validation and standardization in order to better understand the nature and the causes of DNA abnormalities in human spermatozoa ,48. In rThe present study suggests a link between DNA fragmentation and oxidative base damage but lipid peroxidation seems to be an independent sperm decay although all of these alterations are linked to oxidative stress. Free radicals-induced sperm damage has been studied extensively and there is evidence showing that a significant proportion of the DNA damage observed in human spermatozoa is oxidative in nature. However, other molecular mechanisms underlying such damage need to be elucidated by further studies that should take into consideration the major role of oxidative stress in causing sperm DNA damage. In addition, the use of antioxidants could have beneficial effects in preventing such damage and ameliorating semen parameters and reproductive outcomes .The authors declare that they have no competing interests.NZ made the work of conception, methodology, design, and acquisition of data and statistics, analysed and interpreted the data, drafted and revised the manuscript, tables and figures, and revised the manuscript. NFC contributed to the conception and design, analysed and interpreted the data, and revised the manuscript. HEE and JG carried out the flow cytometry analysis and participated in data interpreting. FBA participated in lipid peroxidation measurement and in statistical analyses. FF and LAK revised the manuscript critically for important intellectual content and final approval. All authors read and approved the final manuscript.


Background and Study objective. Focused lung ultrasound (LUS) examinations are important tools in critical care medicine. There is evidence that LUS can be used for the detection of acute thoracic lesions. However, no validated training method is available. The goal of this study was to develop and assess an objective structured clinical examination (OSCE) curriculum for focused thorax, trachea, and lung ultrasound in emergency and critical care medicine (THOLUUSE). Methods. 39 trainees underwent a one-day training course in a prospective educational study, including lectures in sonoanatomy and -pathology of the thorax, case presentations, and hands-on training. Trainees' pre- and posttest performances were assessed by multiple choice questionnaires, visual perception tests by interpretation video clips, practical performance of LUS, and identification of specific ultrasound findings. Results. Trainees postcourse scores of correct MCQ answers increased from 56 ± 4% to 82 ± 2% ; visual perception skills increased from 54 ± 5% to 78 ± 3% (P < 0.001); practical ultrasound skills improved, and correct LUS was performed in 94%. Subgroup analysis revealed that learning success was independent from the trainees' previous ultrasound experience. Conclusions. THOLUUSE significantly improves theoretical and practical skills for the diagnosis of acute thoracic lesions. We propose to implement THOLUUSE in emergency medicine training. Focused lung ultrasound (LUS) examinations are increasingly important diagnostic tools in emergency and critical care medicine –4. A broHowever, unlike focused assessment with sonography in blunt trauma (FAST) that has become a standard procedure, ultrasound examination of thorax and lungs has only recently been established for workup of critically ill patients . Still, While the detection of fluid such as in hematothorax or pleural effusion (PLE) has been described as relatively simple  and ultrTo our knowledge, no focused training method for chest sonography, particularly for the use in emergency situations, has been validated until today.Therefore, we designed an objective structured clinical examination (OSCE)  trainingn = 3), internists (n = 3), cardiologists (n = 4), anesthesiologists (n = 2), and trauma surgeons (n = 2). Medical interns (n = 3) were specifically trained to give instructions for use of ultrasound phantoms. A formal institutional review board approval was obtained at the University of Frankfurt, Medical faculty. All trainees gave informed consent for anonymous analysis of their test results. Patients or relatives of ventilated patients gave their informed consent to serve for teaching purposes according to local ethical standards at our institution.We performed a prospective educational study with a standardized OSCE curriculum. A total of 54 trainees were enrolled into the program in 4 independent training days. Group A included fourteen medical students (age 25 ± 2 years (mean ± standard deviation)) and group B thirty-two anesthesiologists (32 ± 5). Both groups had none or very limited ultrasound knowledge. Group C consisted of eight trauma surgeons (37 ± 6), who all previously underwent a FAST training . InstrucTheoretical training was scheduled for 2.5 hours and included six brief lectures of anatomy, physiology, and pathology of the thorax, as well as four case presentations on clinical scenarios related to PLE and PTX . PracticIn order to support the trainees active learning process, we included custom made gel phantoms into the HOTs. Gel was made by commercially available pork skin leafs . Phantoms were prepared with 20 grams  of gelling leafs per half a litre of distilled water, carefully heated to 60°C, followed by stirring for 1 min. Next, gels were casted into plastic containers (95 × 15 × 15 cm) and allowed to cool down for two hours at room temperature. While still viscous, water and content filled rubber balloons were carefully submerged in the gel body by avoiding insertion of air bubbles. Next, gels were incubated in a refrigerator overnight until solid. Stiff gel phantoms were used without antimicrobiological additives for 10 days. In HOT-1, one station contained a series of rubber balloons. Each balloon was filled with specific contents empirically chosen to mimic typical phenomena found in chest ultrasound: (a) pure water to visualize transmission and reflexion of ultrasound on interfaces with different acoustic impedance, for example, thorax wall, pleural line, and PLE; (b) air to mimic reverberation artifacts found in PTX; (c) parboiled rice grains and starch to mimic partially consolidated PLE and fibrinous structures; (d) olives as substitute for soft but solid tissues; (e) a stone as substitute for carbon-rich structures such as bones with complete ultrasound absorption and dorsal extinction. Finally, balloons with small jelly babies sized less than 1 cm and a tiger duck made of wood of a size of about 2 cm were included as target structures and to practice recognition of specific objects Figures . In HOT-http://dx.doi.org/10.1155/2013/312758), each loop had a duration of 10 seconds followed by a 5-second break to note the results onto a standardized answering form. A self-running DVD was produced with MAGIX 5.5 deluxe . The DVD was started once and ran continuously without replay or break until all video clips were shown. The order of the clips, questions, and answers for postcourse testing was changed in a randomized fashion to prevent memory of order or answers. Finally, at the end of HOT-2, there was (c) a practical postcourse examination in which trainees had to demonstrate their skills in correct positioning of the ultrasound probe and to visualize and identify sixteen predefined sonoanatomic items considered particularly relevant to chest ultrasound (see Appendix Table 2). Over 15 min, trainees performance was observed by an instructor and rated on a standardized score sheet. Instructors were not allowed to help or modify the positioning of the probe, and noted results were blinded to the participants. A cut-off level of 60% correctly performed tasks/identified structures was arbitrarily defined to passing before the onset of the study.For scientific evaluation of the THOLUUSE program, trainees had to pass three types of tests: (a) a theoretical test with multiple-choice questions (MCQs) before (precourse) and after (postcourse) completion of the program. Each MCQ test set contained fifteen textural questions and five questions with scanned ultrasound pictures where specific sonoanatomical structures had to be identified. The maximum time to answer each question was 60 seconds. As one of the posttests, (b) a recognition quiz to test the visual perception skill with fifteen video clips  was applied. Originally, we had scheduled a precourse test for practical skills as well. However, we had to omit precourse testing because of insufficient knowledge and practical skills of most participants before entering the study. Therefore results of the postcourse test were computed against zero.Our null hypothesis was that training would not significantly increase trainees skills. A finding was considered significant when error probability was less than 5% and null hypothesis could be rejected. Results are given as box plots, scatter plots, or mean and confidence intervals or standard deviation if not indicated otherwise. For comparison of the training effects of the different test groups, nonparametric Wilcoxon matched-pairs signed rank test (within groups) or Mann Whitney Test results are shown in two ways: (a) mean result of all trainees passing a test was regarded as general learning effect by examinees and (b) number of trainees who scored positive for a specific question or observation. The latter was interpreted as learning success on a specific item. Statistical analysis and figures were produced with GraphPad Prism software .P < 0.001 for comparison to precourse results), B: 17.1 , and C: 16.8, , respectively , anaesthesiologist residents (group B), and trauma surgeons (group C) mean precourse scores of correct answers were 11.2 , 11.5 (9.8–13.1), and 11.8 (9.5–14.0), respectively. After completion of postcourse exams, all groups of trainees achieved comparable increases in their scores: A: 16.1 , in B from 8.5 (6.7–9.2) to 12.3 , and in C from 9.1 (7.1–11.2) to 12.6 , respectively  to 11.6 , a mean of 95% of the postcourse sonograms was conducted in a technically correct fashion .Subgroup analysis of practical performance skills revealed no statistical significant differences of visualization and interpretation of distinct anatomic sites/learning items .In the present study, we developed and assessed an OSCE-based training program for THOLUUSE. For more than a decade, FAST has been established as a standard procedure in shock rooms or emergency departments for early diagnosis of cavity bleeding in blunt trauma. Kirkpatrick et al.  suggesteIt has been questioned if a one-day training course with a brief factual input and few essential exams can teach ultrasound skills that can be of practical relevance for the trainee . It is wOur results show that upon completion of a one-day LUS course participants were able to perform a standardized sonographic examination of the thorax and correctly identify and interpret the most relevant physiological and pathological findings, including PLE and PTX. Of note, we found no significant differences between medical students and postgraduates from different medical professions, and trainees postcourse performances were independent of previous levels of ultrasound experience. Our findings are supported by a project for experimental ultrasound learning, the Advanced Diagnostic Ultrasound in Microgravity (ADUM) , launcheA limitation of this study was that postcourse performance of trainees could only be assessed shortly after completion of the program. Previous evaluations of FAST course programs demonstrated that trainees skills decreased as a function of time, particularly when ultrasound techniques were not practiced on a regular basis and further supervised teaching was not available . TherefoIn this respect, the THOLUUSE concept proposed in this study was certified by the German Society of Ultrasound in Medicine (DEGUM) and incorporated substantially into a modular blended learning programme of the German Society of Anesthesiology and Intensive Care Medicine (DGAI) for official training in emergency and critical care medicine .The results of our study demonstrate that a one-day training program like THOLUUSE significantly improves theoretical and practical skills for sonographic diagnosis of acute thoracic lesions, including PLE and PTX. Postcourse gain of competence was independent of previous ultrasound expertise of the trainees, and we propose to implement THOLUUSE in the training of medical emergencies. THOLUUSE long time impact on the management of patients in emergency and critical care medicine needs further investigation.Showing the pre- and post-course video simulation test that was used to assess visual perceptive skills and a list of all items the trainees had to identify during the post-course examination.Click here for additional data file.


The mechanism of the interaction was further elucidated in vitro. Metformin did not suppress the growth of already established tumors, nor did metformin augment tumor shrinkage by gefitinib. However, metformin significantly suppressed the regrowth of the tumor after effective treatment with gefitinib, suggesting the specific effect of metformin on the residual cells. Cytotoxicity of metformin was characterized by the absence of apoptosis induction and unremarkable cell cycle shift in vitro. The residual cell population after treatment with gefitinib was characterized by enriched cells with high expression of CD133 and CD24. Metformin was still effective on this specific cell population. Targeting residual cells after chemotherapy may represent an effective novel strategy for the treatment of cancer. Elucidating the mechanism of metformin cytotoxicity provides insights into future development of anticancer therapeutics.Cancer chemotherapy, including molecular targeted therapy, has major limitations because it does not kill all the cancer cells; the residual cells survive until they acquire chemoresistance. In the present study, the combined effects of metformin and gefitinib were examined  EGFR) mutation is treated by EGFR-tyrosine kinase inhibitors (TKIs)  . Thus, es (TKIs) . The secs) , an enzyme that plays an important role in insulin signaling, whole body energy balance and the metabolism of glucose and fats, resulting in lowering of blood glucose . Metform cancers –20. Alth cancers ,22 and m cancers . Second, cancers ,25 and t cancers . Notably cancers . However cancers ,17 or no cancers ,16, or w cancers ,18,20,27 cancers ,29.in vivo experiments suggested a unique anticancer action for metformin, specifically on residual cells after chemotherapy. The mechanism was further elucidated with a series of in vitro experiments.In the present study, EGFR gene (del E746–A750) in exon 19  was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C until use. Metformin  was dissolved in phosphate buffered saline (PBS) at a concentration of 100 mM and stored at 4°C. A cisplatin solution at a concentration of 0.5 mg/ml (pH 2.5–5.5) was purchased from Nihon Kayaku . Each drug was diluted in the complete medium for each experiment and the final concentration of DMSO was <0.1%.A human lung adenocarcinoma cell line, PC9, purchased from Riken Cell Bank , was used throughout the study. This cell line has an activating deletion of the  exon 19 . The cel6 cells per mouse) were injected subcutaneously into their flanks. At day 16 (when the tumor volumes had reached ∼300 mm3), the mice were randomly allocated into 4 groups (7 mice per group). In every group, administration of either saline alone or gefitinib suspended in saline  and either PBS alone or metformin dissolved in PBS  were started. Either saline alone or gefitinib suspended in saline was continued for 14 days and either PBS alone or metformin dissolved in PBS was continued until terminating observation. In the first group, only saline (p.o.) and PBS (i.p.) were administered (control). In the second group, metformin dissolved in PBS was administered. In the third group, gefitinib suspended in saline was administered. In the fourth group, both gefitinib and metformin were administered 3/2, where A and B represent the larger and smaller diameters of the tumor, respectively. The tumor size was measured twice a week during the observation period. The animal experiments were approved by the animal ethics review board of Chiba University (protocol no. A22-186) and were conducted in an animal facility at Chiba University under the strict SPF conditions in accordance with the established institutional guidelines.Five to 6-week-old female severe combined immunodeficient (SCID) mice were acclimatized to local conditions for a week before starting the experiments. Aliquots of the cell suspension  was calculated. A series of preliminary experiments indicated that metformin at 10 mM, gefitinib at 0.03 μM and cisplatin at 1.5 μg/ml were nearly equivalent in reducing cell numbers to 10% of the cell number obtained without adding the agents (control) at 72 h  and examined by fluorescence microscopy according to the manufacturer's instructions. Cells with aggregated or fragmented chromatin were regarded as apoptotic cells and 500 cells in each experiment were counted to calculate the apoptotic cell ratio. To determine the caspase activity in the cell extracts, a colorimetric assay was used to monitor the absorbance at 405 nm of p-nitroanilide (pNA) released from the synthetic substrates. Caspase 3 and 8 activities were evaluated with synthetic substrates DEVD-pNA and IETD-pNA, respectively, using the colorimetric assay kit APOPCYTO  according to the manufacturer's instructions. In each assay, ∼200 μg of protein was extracted from the cells treated with each agent for 24 h.Apoptosis was evaluated by Hoechst staining and caspase activity determination. For Hoechst staining, cells treated with agents for 48 h were trypsinized and harvested together with the floating cells, fixed with 70% ethanol, stained with 2 μl of PI/RNase staining buffer (BD Biosciences) and incubated for 15 min at room temperature.Cell cycle distributions were determined by a propidium iodide single-color flow cytometric method , according to the manufacturer's instructions. Briefly, the cells were trypsinized, washed twice with ice-cold PBS, fixed with 70% ethanol and then stored at −20°C until analysis. Before analysis by FACS and CellQuest software (BD Biosciences), the cell suspensions were washed twice with PBS, suspended in 500 The cells were cultured in a chamber slide for 24 h, followed by treatment with the agents for 24 h. After removal of the medium containing the agents, the cells were fixed in a 1:1 mixture of methanol and acetone for 2 min, followed by blocking with normal goat serum for 30 min. The cells were incubated with primary anti-human CD133 antibodies  overnight at 4°C. The cells were then washed 3 times in PBS and incubated with the secondary antibody (anti-mouse IgG) conjugated to the Alexa488 fluorescent dye for 1 h at room temperature. The stained cells were embedded in VectaShield mounting medium with DAPI  and were examined with a Nikon Eclipse 80i microscope  using the VB-7210 imaging system . Staining results were directly observed at a magnification of ×200 using a fluorescence microscope.6) in a single-cell suspension were resuspended in the staining buffer (PBS containing 2% FBS) and labeled with the antibodies, followed by washing and resuspension in 500 μl of staining buffer according to the manufacturer's instructions. The cells were also labeled with propidium iodide to enrich viable cells and analyzed with a JSAN cell sorter and AppSan software .For analysis of cell-surface marker expression by FACS, anti-human CD24 antibodies conjugated to phycoerythrin  and anti-human CD44 antibodies conjugated with allophycocyanin  were used. The cells treated with the agents for 24 h were trypsinized and washed 3 times with PBS. The cells  was considered statistically significant.3, metformin did not reduce tumor growth  was enriched to 81.8±12.1% (n=3). The al cells .EGFR exon 19 deletion mutation that renders EGFR sensitive to the TKIs. In vivo experiments with xenografts derived from these cells resulted in the following observations: i) metformin exerted no effect on already grown tumors (>300 mm3), ii) metformin had no additional effect on tumor shrinkage by gefitinib, iii) the tumors regrew after withdrawing gefitinib even after the treatment had resulted in complete regression of the tumors and iv) metformin significantly suppressed the regrowth of the tumors after withdrawing gefitinib treatment. These observations suggest that metformin is effective specifically on residual cells after gefitinib treatment; however, metformin is not sufficiently effective to suppress growth of already established tumors.The present study employed the human lung adenocarcinoma cell line PC9, which possesses an in vitro experiments were conducted. Cisplatin was included as a positive control in some of the experiments. A dose that resulted in an in vitro cell number reduction to 10% of the original cell number was chosen for each agent. Metformin did not induce apoptosis when assessed by Hoechst staining and caspase 3 and 8 activity determination, in contrast to gefitinib and cisplatin. Moreover, apoptosis induction by metformin treatment was significantly lower, even lower than what was observed in the control experiments, suggesting an apoptosis-protective property of metformin. This is consistent with a previous report that demonstrated a preventive effect of AMPK on apoptosis  was obtained by FACS sorting to directly examine chemosensitivity. These cells were slightly but significantly more resistant to gefitinib than the parental cells, whereas their sensitivity to metformin was identical to the parental cells, suggesting that metformin was effective against residual cells after gefitinib treatment. Nevertheless, the degree of augmented resistance to gefitinib in cells with CD24 expression was unexpectedly small. This can be explained if the resistant cells express CD24 and if only a part of the cell population with CD24 expression is resistant to gefitinib.Based on the reported drug resistance of cancer stem cells  and the s report , suggestThe nature and properties of the residual cells after treatment with gefitinib are unclear. Although CD133 and CD24 are putative markers for cancer stem cells in human brain and colon cancers, respectively, the present results do not indicate that the residual cells are cancer stem cells because cancer stem cells in human lung cancer have not yet been identified. Considering their prompt emergence in a short period, non-mutational mechanisms, including epigenetic change and selection of resistant cells from a heterogeneous cell population, seem to be the most likely routes of chemo-resistance. Cancer stem cells and epithelial-mesenchymal transitions might be possible candidates for selection. Specific targeting of residual cells after chemotherapy may be a suitable approach to cure cancers. The present study highlighted metformin as a candidate for targeting residual cells and we envision that further elucidation of the detailed molecular mechanism of the cytotoxicity of metformin represents progress in cancer therapy.


Bombyx mori L. (Lepidoptera: Bombycidae) were studied. The results indicated that food ingestion and assimilation were significantly higher among silkworm batches where optimum temperature and humidity were maintained compared with silkworm batches exposed to natural climatic conditions of the respective season. However, during summer the nutritional efficiency parameters were significantly higher among silkworms reared under natural temperature and humidity conditions when compared with the control. During the winter and rainy season, the nutritional efficiency parameters were significantly higher in control batches, where optimum temperature and humidity were maintained. Ingesta and digesta required to produce one gram of cocoon/shell were also lower in control batches for all seasons except summer. This may be due to the physiological adaptation of silkworms to overcome stress during the summer season.Food consumption and utilization is influenced by various biotic and abiotic factors. Under different environmental, feeding, and nutritional conditions, and with ingestion of the same amount of mulberry leaves, the silkworm shows significant difference in its ability to digest, absorb, and convert food to body matter. Here, influences of season, temperature, and humidity on food intake, assimilation, and conversion efficiency of the Indian bivoltine hybrid (CSR2 × CSR4)  Bombyx mori L. (Lepidoptera: Bombycidae) is a Poikilothermic insect and the main source for the production of silk. Environmental factors such as temperature, humidity, light, air, and feed quality and quantity have intimate influences on its growth and development. Food consumption and utilization are influenced by various biotic and abiotic factors  up to fourth molt.A productive and popular bivoltine hybrid silkworm (CSR2 × CSR4) developed by breeders of Central Sericultural Research and Training Institute, Mysore, India under the collaboration of Japanese subject experts was used in this experiment. The race is suitable to rear during more favorable seasons (August-February) under Indian environmental conditions. This hybrid is popular for its high survivability, high yield, high silk ratio, and its production of quality bivoltine silk matching international standards. Young silkworm rearing was conducted by following the new standard rearing package and practices  by provith instar larvae, as 80–85% of the total leaf is consumed in this stage. The experiment was conducted during different seasons including summer, rainy, and winter. After the resumption from 4th molt, 50 larvae each for three replications were separated and reared in natural conditions without maintaining temperature and humidity, and exposed to extreme temperature variation and high humidity conditions of the different seasons. For every season, a control batch with three replications of 50 larvae each was separated and reared in a sericatron—an environmental chamber with precise and automatic control facilities for uniform maintenance of temperature and humidity—and maintained at a temperature of 25 ± 1 °C and 70 ± 5% humidity. Known quantities of fresh quality mulberry leaves were provided to silkworms three times and followed the standard recommendation of the bivoltine rearing package , reference ratio (RR), consumption index (CI), conversion of ingesta and digesta (ECI and ECD) to larval body, cocoon, shell, and ingesta and digesta required to produce 1 g of cocoon and shell were determined by the standard gravimetric method described by Waldbauer Approximate digestibility percentage (AD%) = (dry weight of food ingested — dry weight of feces / dry weight of food ingested) × 100Reference ratio = dry weight of food ingested / dry weight of excretaECI/ECD to larva (%) = (dry weight gained by larvae during feeding period / dry weight of ingesta or digesta) × 100ECI/ECD to cocoon (%) = (dry weight of cocoon / dry weight of food ingested or digested) × 100ECI/ECD to cocoon shell (%) = (dry weight of cocoon shell / dry weight of food ingested or digested) × 100Ingesta required to produce 1 g of cocoon / cocoon shell (ingesta / g cocoon / shell) = dry weight of ingesta / dry cocoon or cocoon shell weightDigesta required producing 1 g of cocoon / cocoon shell (digesta / g cocoon / shell) = dry weight of digesta / dry cocoon or cocoon shell weightVarious nutritional indices are calculated and presented in Similar to ingesta, digesta was also significantly higher in control batches when compared with respective natural temperature and humidity maintained batches. In summer, the lowest value of digesta was recorded in T1 (1.202 g) when compared with respective control (C1: 1.595 g). Significantly lower digesta was recorded in rainy (1.721 g) and winter (1.702 g) seasons when compared with respective control. Analysis by season showed that the maximum value of digesta was recorded during winter (T2: 1.721 g), and the lowest value was recorded in the summer (T1 : 1.202 g). Significantly lower approximate digestibility was recorded in summer (T1: 26.22%) when compared to respective control (C1: 33.25%). In other seasons, the same trend was observed, as significantly lower approximate digestibility was recorded in rainy (T2: 36.04%) and winter (T3: 35.66%) seasons when compared with respective control.Significantly higher reference ratio (RR) values were recorded in control batches when compared with the seasonal batches . Significantly higher consumption indices were recorded in control batches , where optimum conditions of temperature and humidity were maintained. Seasonal variation data indicated that during summer, the lowest consumption index was recorded (T1: 0.392).Various nutritional efficiency parameters were studied and results are presented in Similarly, ECD—larva was recorded as significantly higher during the summer season (T1: 68.88%) when compared with other two seasons. A significant difference of ECD—larva between control and normal batches was recorded during all seasons. ECD—larva (T1: 68.88%) was significantly higher when compared with control (C1: 62.88%) batch of summer. Significantly, lower values of ECD—larva, 61.57 and 61.82%, were recorded for rainy and winter seasons, respectively.Significant difference of ECI—cocoon was observed between control and batches reared under natural climatic conditions. However, ECI—cocoon was significantly higher in summer—reared batches (18.05%) when compared to summer control (C1: 17.52%). In the rainy season, a significantly higher ECI—cocoon value of 17.32% was recorded for control batches when compared with the treated batches. Similarly, in winter, a significantly higher ECI—cocoon value (C3: 17.08%) was reported for control batches when compared with the treated batch (T3: 16.45%).Similarly, ECD—cocoon was significantly higher in summer—reared batches (T1: 66.25%) when compared with the control (C1: 62.51%) and batches reared in the other two seasons. However, in the rainy and winter seasons, significantly higher ECD—cocoon was recorded in control  when compared with batches reared under natural conditions  in the rainy and winter seasons, respectively.Significantly higher ECI—shell was recorded in batches reared under natural conditions of summer (T1: 8.91%) when compared with the control (8.12%) and batches reared in other seasons. However, in the rainy and winter seasons, significantly lower ECI—shell was recorded for batches reared under natural conditions . Significant variation of ECD—shell was recorded among batches reared in different seasons. Similarly, higher ECD—shell (T1: 30.05%) was recorded for summer—reared batches when compared with batches of other seasons. In rainy and winter seasons, significantly lower ECD—shell was recorded in batches reared under natural conditions .Ingesta required to produce 1 g of cocoon was recorded significantly lower (5.42 g) in summer—reared batches when compared with corresponding control (5.66 g). Values of 5.78 and 5.82 g were recorded for rainy and winter seasons, respectively. Seasonal variation of data showed that in the summer season, the amount of food required to produce 1 g of cocoon was less than the other two seasons .The same trend was noticed in digesta required to produce 1 g of cocoon (D/g cocoon). This value was significantly lower in summer natural condition reared batches (T1: 1.59 g) when compared with the control (C1: 1.72 g), and it varied significantly when compared with other two seasons .Ingesta required to produce 1 g of shell (I/g shell) was also significantly lower in summer natural condition reared batches (T1: 10.89 g) when compared with the summer control (C1: 11.25 g) and with batches of the other two seasons . However, in the winter and rainy seasons, control batches had significantly lower I/g shell values.The same trend was noticed in the case of D/g shell, with lower values for batches exposed to summer climatic conditions (T1: 3.28 g) when compared with summer control (C1: 3.33 g) and other batches. However, in the other two seasons, significantly higher D/g shell values were recorded in control batches for each respective season .th instar) in the bivoltine race, resulting in a shorter larval duration, adversely affected larval weight and other correlated traits.Most of the strains of bivoltine silkworm, due to consistent domestication, have become highly sensitive to variations in environmental conditions, especially seasonal variations in temperature and humidity in tropical parts of the Indian subcontinent. Fifth instar larval duration was significantly lower in the summer under natural environmental conditions compared with the control, where optimum temperature and humidity were maintained. Retarded growth in insects may be due not only to the nutritional inadequacy of the diet, but also due to the effect of various environmental conditions. Growth also depends on the digestion and utilization of food, which varies from species to species and even between different sexes of the same species. Further, reduction in feeding time in the most active feeding stage  were observed in all the control batches when compared with the respective batches reared in different seasons. Seasonal variation in AD% showed that winter season performance was comparatively better with a higher digestibility percentage. Higher values of AD% in control batches indicated the greater suitability of mulberry leaves and environmental conditions provided to silkworms. Since the digestibility differs in different treatments, the proportion of food intake and production of fecal matter also varied . Fecal mReference ratio (RR) is an indirect expression of absorption and assimilation of food. Higher RR values indicate high rate of digestion and absorption of food. The study results also found that comparatively higher RR values were recorded in control batches, and seasonal analysis shows that the maximum and minimum RR values were recorded in winter and summer, respectively. The consumption index (CI) is defined as the feeding rate at which nutrients enter into the digestive system. Trivedy and Nair  suggesteIn a general entomological scenario, the nutritional efficiency of an insect may be considered an important parameter. In sericulture, however, nutritional efficiency becomes something of industrial importance. The dietary efficiency of silkworms can contribute considerably to the cost—benefit ratio of sericultural practice up to the level of cocoon production. The share of mulberry leaves accounts for more than 50% of the totalcost of silkworm rearing. Different strains and hybrids of silkworm show significant differences in ability to digest, absorb, and convert mulberry leaves to body substance, and subsequently to the primary marketable product, the cocoon. The effect of different rearing seasons, planting density, rearing techniques, rearing conditions, quality of leaves, feeding proportion, and various chemicals such as food additives, vitamins, antibiotics, and hormones have an influence on the efficiency of conversion of food. In China, sericulture scientists have realized the importance of dietary efficiency, and they are engaged in finding effective ways to enhance leaf—silk conversion rate. This can be improved by breeding desirable silkworm strains and mulberry genotypes, providing good rearing environment and nutrition level, and by applying some insect growth regulators.It is well known that food intake and silk production in silkworms is closely related to nutritional factors. Dietary efficiency of silkworms plays a major role in converting consumed mulberry leaves to silk. Efficiency of conversion of ingested (ECI) mulberry leaves into silk or leaf silk conversion rate is a better economic index in cocoon production. Trivedi and Nair  also supIn the present study, the feed conversion efficiency parameters such as ECI and ECD to larva, cocoon, and shell were significantly higher in control batches of the different seasons, except for summer, where all efficiency parameters connected with ingestion and digestion were significantly higher in batches exposed to natural weather conditions.Many insects have adaptive behaviors to cope with stressful environmental conditions such as those present in the summer months; most of the feed conversion efficiency parameters were recorded significantly higher during this time period. Insects have evolved a variety of strategies to acquire and accumulate energy from nutrients and water available from food in a given environmental condition . Singh aIngesta required to produce 1 g of cocoon was lower in summer—reared batches when compared with the corresponding control. This indicated that during the summer environmental conditions were unfavorable for silkworm growth, larval duration was shorter, the lifecycle was completed early and naturally, and less food was required to produce 1 g of cocoon. However, in the other two seasons, optimum temperature and humidity maintained (control) batches had lower I/g cocoon values when compared with batches reared under natural climatic conditions. In summer— reared batches, the lower ingesta and digesta required to produce 1 g cocoon and cocoon shell was due to its efficiency in converting the food under stressful conditions, which also depends on the genotypic character and its interaction with biotic and abiotic factors . In the Results show that the food ingestion and assimilation was significantly higher in optimum temperature and humidity maintained silkworm batches when compared with silkworm batches exposed to natural climatic conditions of respective seasons. However, nutritional efficiency parameters were higher in batches exposed to natural conditions of summer when compared with the control. During the winter and rainy seasons, nutritional efficiency parameters were significantly higher in control batches where optimum temperature and humidity were maintained. Ingesta and digesta required to produce 1 g of cocoon/shell were also recorded lower in control batches of all seasons except summer, which may be due to the physiological adaptation to overcome stress during the summer season.For the last two decades, the introduction of productive bivoltine hybrids and rearing management in India has resulted in a phenomenal increase in yield and income level of the sericulture farmers, and has increased output of quality raw silk. However, these productive bivoltine hybrids can only provide large benefits to farmers who are able to provide high input and managerial skills. These hybrids lack genetic plasticity to cope with adverse temperature and humidity conditions of India. Therefore, it has become highly imperative to develop silkworm breeds tolerant to diseases and adverse climatic condition, or plan effective strategies for management of climatic conditions during silkworm rearing for better silk production.


Defects in the ubiquitin-proteasome system have been related to aging and the development of neurodegenerative disease, although the effects of deficient proteasome activity during early postnatal development are poorly understood. Accordingly, we have assessed how proteasome dysfunction during early postnatal development, induced by administering proteasome inhibitors daily during the first 10 days of life, affects the behaviour of adult mice. We found that this regime of exposure to the proteasome inhibitors MG132 or lactacystin did not produce significant behavioural or morphological changes in the first 15 days of life. However, towards the end of the treatment with proteasome inhibitors, there was a loss of mitochondrial markers and activity, and an increase in DNA oxidation. On reaching adulthood, the memory of mice that were injected with proteasome inhibitors postnatally was impaired in hippocampal and amygdala-dependent tasks, and they suffered motor dysfunction and imbalance. These behavioural deficiencies were correlated with neuronal loss in the hippocampus, amygdala and brainstem, and with diminished adult neurogenesis. Accordingly, impairing proteasome activity at early postnatal ages appears to cause morphological and behavioural alterations in adult mice that resemble those associated with certain neurodegenerative diseases and/or syndromes of mental retardation. In the rodent hippocampus, many of these connections reach maturity by the end of the second postnatal week, following an identifiable developmental progression that appears to be common to a wide variety of species Proteins are the motors of virtually all biological processes, and the finely tuned equilibrium between their synthesis and degradation influences cellular homoeostasis. Accordingly, the deregulation of protein clearance and synthesis contributes to cell senescence, aging and various age-related disorders in the central nervous system In the last decade, considerable research has focused on the role of the UPS in aging, neurodegenerative diseases and synaptic plasticity. Here we focus on the role of the UPS during early postnatal mouse development and on the consequences of postnatal proteasome inhibition in later life. To address these issues, we administrated proteasome inhibitors in the temporal window from 1 to 10 days life as in this period the blood brain barrier is not completely closed and as such, proteasome inhibitors can reach the brain easily. Subchronic administration of the proteasome inhibitors MG132 or lactacystin provoked a significant decrease in chymotrypsin activity, an accumulation of ubiquitinated proteins, a decrease in mitochondrial markers and of their activity, and an increase in DNA oxidation. However, proteasome inhibition caused no behavioural or morphological alterations within this timeframe. Nevertheless, upon reaching 3–5 months of age, mice treated postnatally with proteasome inhibitors displayed impaired hippocampal- and amygdala-cognition, and mild motor dysfunction. These behavioural deficiencies were correlated with neuronal loss in the hippocampus, amygdala and brainstem, as well as diminished neurogenesis. Accordingly, these results indicate that decreased UPS function during early postnatal development provokes an acceleration of several features associated with aging, including neuronal degeneration, as well as motor and cognitive impairment, inducing a phenotype similar to that found in mouse models of mental retardation.p = 0.003; Lactacystin: t(6) = 4.605, p = 0.004. p<0.001; Lactacystin: t(9) = 2.814, p = 0.002. To study the effect of early postnatal proteasome inhibition on adult mice during the first 10 days of life, we administered daily systemic injections of a proteasome inhibitor, MG132 (2.5 mg/kg) or lactacystin (1 mg/kg). To quantify the effect of proteasome inhibition on ubiquitinated proteins, western blots of total brain protein from PD10 mice were probed with an antibody against ubiquitin. Ubiquitinated proteins were more abundant in brain extracts from MG132- and lactacystin-treated mice than in their respective controls (MG132: t(6) = 4.679, p = 0.002. p = 0.001. p = 0.02. We investigated the effects of proteasome inhibition on other molecular processes, focusing initially on the mitochondria as there is evidence that mitochondrial quality is controlled by the ubiquitin-proteasome system Fig. S1). In terms of balance reflexes  or locomotor activity (pivoting activity test: ), there were no significant differences between mice injected with proteasome inhibitors and control mice during the first two weeks of life. Moreover, while some differences in the body weight of MG132-treated mice were observed initially , these disappeared later with age. When brain morphology was analyzed by staining with antibodies against calbindin, tyrosine hydroxylase and doublecortin, there were no significant differences in the hippocampus, cerebellum, amygdala or substantia nigra of PD15 mice treated with proteasome inhibitors when compared to their control littermates .To determine the effect of proteasome inhibition on the postnatal psychomotor development of mice, we performed behavioural tests included in the Fox's battery daily from PD 1 to 15  = 0.032, p = 2.26). These findings suggest that proteasome inhibition during early postnatal development provokes cognitive deficiencies in adulthood.To determine whether temporary inhibition of proteasome activity during the first 10 days of life produces behavioural alterations in adult mice, exploratory activity in 3–5 month old mice was evaluated over 5 minutes in the open field test. Mice treated with proteasome inhibitors exhibited a mild non-significant decrease in locomotor activity when compared with control mice Fig. 3A.). No significant differences were found in vehicle-, MG132- and lactacystin-injected mice in any of these tests. Furthermore, when we studied the histology of the spinal cord, there were no differences between these three experimental groups . Hence, mice treated with proteasome inhibitors during postnatal development appear to suffer depression and altered motor coordination in adulthood.To study cognition in adult mice subjected to postnatal proteasome inhibition, 3–5 month old mice were subjected to tests of anxiety-like behaviour in the elevated plus maze, which revealed no differences between mice treated with proteasome inhibitors and their controls Fig. 4A.+ neurons were detected in the SN and VTA of adult mice treated postnatally with lactacystin than in control mice (SN: t (15) = 4.351, p<0.001; VTA: t (15) = 6.846, p<0.001. + neurons in the VTA (t (15) = 3.954, p<0.001). These decreases in the number of TH+ neurons in the SN and VTA were probably correlated with the severity of the impairments in their co-ordination.Given the role of brainstem, substantia nigra (SN) and ventral tegmental area (VTA) dopaminergic neurons in regulating movement co-ordination, we assessed the levels of tyrosine hydroxylase (TH) protein by immunohistochemistry. Fewer THp<0.001. p = 0.006. p = 0.49). Furthermore, proteasome inhibitor treatment failed to alter the exploration times with respect to the vehicle treated group in the OR test (p = 0.92. p = 0.01).To identify possible cognitive deficiencies in adult mice treated postnatally with proteasome inhibitors, as suggested by the exploratory habituation findings, we performed several learning and memory tests that depend on different brain areas. In the hippocampal-dependent Y-maze, we evaluated the preference of the mice for the novel arm of the maze. Animals treated with proteasome inhibitors at early ages showed a weaker preference for the novel arm than control mice  = 11.46,  OR test . Finally and As the hippocampus and amygdala play central roles in learning acquisition and memory consolidation in the tests used, these structures were analysed morphologically by calbindin immunohistochemistry. In both structures, fewer neurons expressing calbindin were observed in adult mice treated postnatally with proteasome inhibitors when compared to the controls  and 8. As aThe UPS provides a useful mechanism to dispose of biologically compromised proteins in a cell, including mutant, misfolded, damaged, terminally modified or over-represented proteins At the cellular level, motor co-ordination and cognitive deficits are linked to functional impairment in specific brain areas. Motor co-ordination is primarily linked to the brainstem, striatum and cerebellum, as evident through the characteristic motor disturbances associated with neurodegeneration in these areas in Parkinson's and Huntington's diseases, and in ataxia The early postnatal period is a critical stage of brain development during which excitatory and inhibitory functions in the brain are organized and mature. For this reason, environmental stress or exposure to excitatory toxins during this period can provoke behavioural alterations in adulthood Proteasome dysfunction is considered a key contributor to aging-associated neurodegeneration. Indeed, brains from aging individuals and/or those suffering neurodegeneration exhibit clear deficiencies in proteasome activity. Furthermore, mutations in some of the components of the proteasome pathways have been linked to neurodegeneration, mental retardation and other neurodevelopmental diseases. Together, our results demonstrate that subchronic proteasome inhibition during early postnatal development, probably through mitochondrial dysfunction, triggers the development of a complex behavioural phenotype that is correlated with degeneration in specific brain areas. This phenotype shares many features with mental retardation syndromes, suggesting that proteasome dysfunction plays a critical role in the development of these syndromes.ad libitum. Breeding pairs were formed and females were isolated when visibly close to parturition. Litters were adjusted to a maximum of ten pups in order to avoid any effects of parity on behavioural ontogeny Swiss mice weighing 25–30 g were obtained from University of Seville animal facilities and used as the parental mice. The mice were maintained under standard housing conditions, at 21±1°C with a photoperiod of 12:12 h (lights on at 07:30 h), and using dust-free sawdust. Food pellets and water were available All pregnant dams were allowed to deliver spontaneously. The day of birth was designated as postnatal day (PD) 0 (±6 h). On delivery, the litter size of each dam was recorded and each pup was checked for gross abnormalities. Pups were individually marked with India ink on PD1 and nursed by their natural dams until weaning. From PD 1 to 10 (inclusive), the pups were weighed and subcutaneously administered proteasome inhibitors daily  or the vehicle alone . After weaning, the mice were allowed to grow to 3–5 months of age in groups of 4–5. Upon reaching adulthood (from 3 to 5 months old), cognitive behavioural tests were performed. The sex distribution of the treatments was random and while histological analysis at P15 was performed mainly on female mice, adult behaviour and histology were performed on male mice in order to avoid the oestrus cycle. All experimental protocols were approved by the Ethics Committee of the Pablo de Olavide University (07/4-20/12/2008), in accordance with the European Community guidelines (86/609/EEC amended by Directive 2005/65/EC) and the Spanish regulations for the procurement and care of experimental animals .During the testing protocols included in the Fox battery Behavioural tests were performed in a room with homogeneous sound and light. To eliminate any odours or traces that might affect the outcome of the test, the behavioural apparatus was cleaned with 70% ethanol (Panreac Química S.A.U) between the testing of each animal. The order in which the tests were run was always the same: Open field, object recognition memory, Y maze, plus maze, tail suspension and step down passive avoidance.To evaluate locomotor and exploratory activity, mice were placed for 5 minutes in an open field (38×21×15 cm) (Cybertec S.A.). This apparatus consisted of a walled platform containing infrared emitters and sensors (IR) coupled to an actimeter, and the movement sensor was connected to a computer that recorded the number of times the mouse interrupted the IR beams/min.The elevated plus maze consisted of two open and two closed arms of equal dimensions extending from a central area, and it was elevated 70 cm above the ground. Mice were placed at the end of the open arm in brightly lit conditions and allowed to explore the maze for 5 minutes. The percentage of entries and the time spent in the open and closed arms were measured.The Y maze used had three equal sized arms (8×40×20 cm), the walls of which were decorated with different patterns to enable the mice to discriminate between the arms. Mice were allowed to freely explore two arms during a 10 minute training session, after which they were returned to their cages for 2 minutes while olfactory cues were cleaned from the maze. Subsequently, mice were returned to the maze and allowed to explore all three arms for 10 minutes. Short-term memory was determined as the number of entries into the new arms/average number of entries into the familiar arms.novel−tfamiliar)/(tnovel+tfamiliar)]. The criteria for exploration were based strictly on active exploration. Exploration of an object was defined as directing the nose toward the object at a distance of ±1.5 cm and/or touching the object with the nose or vibrissae. Circling or sitting on the object were not considered exploratory behaviour. All trials were performed by an experimenter blind to the drug treatments.Mice were tested in a rectangular arena (55×40×40 cm) located in a room with dim lighting and constant background noise. For the object recognition protocol, two different objects were placed in the arena during the training phase. The animal's memory of the original object was assessed by comparing the amount of time spent exploring the novel object against that for the familiar one. The objects consisted of plastic pieces of different shapes and they were cleaned thoroughly with 70% ethanol between trials to remove any olfactory cues. Before the experiment, mice were habituated to the arena for 20 min in the absence of the objects on 2 consecutive days. On the day of training, mice were allowed 15 min to explore the two objects. Retention tests were then performed at the times indicated after the training session by placing the mice back in the arena for a 10 min session after randomly exchanging one of the familiar objects for a novel object. The time spent exploring each object was recorded and the relative exploration of the novel object was expressed by a discrimination index , which was subsequently reversed. B–E, Fox Battery tests carried out in animals treated with proteasome inhibitors revealed no significant differences with respect to the group that received the vehicle alone in terms of the righting reflex (B), negative geotaxia (C), rotating activity (D) or muscle strength in the suspension test (E). Taken together these data, suggest that proteasome inhibition during early postnatal development does not affect the pup's behaviour.(TIF)Click here for additional data file.Figure S2Postnatal proteasome treatment caused no morphological alterations at PD15. At PD15, mice treated with proteasome inhibitors or the vehicle alone were sacrificed, and their brain histology was analysed. A–C Immunohistochemistry for calbindin revealed no differences in hippocampal dendritic morphology (A) in either the cerebellum (B) or amygdala (C) of mice treated with proteasome inhibitors, or with the vehicle alone. D Tyrosine hydroxylase immunohistochemistry revealed no differences in neuronal density in the substantia nigra of mice injected with proteasome inhibitors or the vehicle alone. E Neurogenesis in P15 mice was unaffected by postnatal proteasome inhibition, as revealed by doublecortin (DCX) immunostaining.(TIF)Click here for additional data file.Figure S3Proteasome inhibition during early postnatal development does not induce motor behaviour or morphological alterations in the spinal cord of adult mice. A–C, Motor function in adult mice that were treated with proteasome inhibitors during early life was measured by their performance in the rotarod (A), treadmill (B) and grip strength (C) tests. D, Representative Nissl staining microphotograph at lumbar 4–5 level of spinal cord is shown (D).(TIF)Click here for additional data file.Table S1Total object exploration times (in seconds) of adult mice injected postnatally with the vehicle alone, MG-132 or lactacystin in each session of a 15-minute OR memory test. STM, short-term memory; LTM, long-term memory.(DOC)Click here for additional data file.Methods S1Detailed description of neonatal behavioural tests included in the Fox battery.(DOC)Click here for additional data file.


Canis lycaon) sampled in Algonquin Provincial Park (APP), Ontario, Canada from 1964 to 2007. Research culls in 1964 and 1965 killed the majority of wolves within a study region of APP, accounting for approximately 36% of the park's wolf population at a time when coyotes were colonizing the region. The culls were followed by a significant decrease in an eastern wolf mitochondrial DNA (mtDNA) haplotype (C1) in the Park's wolf population, as well as an increase in coyote mitochondrial and nuclear DNA. The introgression of nuclear DNA from coyotes, however, appears to have been curtailed by legislation that extended wolf protection outside park boundaries in 2001, although eastern wolf mtDNA haplotype C1 continued to decline and is now rare within the park population. We conclude that the wolf culls transformed the genetic composition of this unique eastern wolf population by facilitating coyote introgression. These results demonstrate that intense localized harvest of a seemingly abundant species can lead to unexpected hybridization events that encumber future conservation efforts. Ultimately, researchers need to contemplate not only the ethics of research methods, but also that future implications may be obscured by gaps in our current scientific understanding.Despite ethical arguments against lethal control of wildlife populations, culling is routinely used for the management of predators, invasive or pest species, and infectious diseases. Here, we demonstrate that culling of wildlife can have unforeseen impacts that can be detrimental to future conservation efforts. Specifically, we analyzed genetic data from eastern wolves ( Although lethal sampling of wildlife for ecological experimentation was common up until the second half of the 20th century, the emergence of a stronger environmental ethic in recent decades has rendered the practice generally indefensible . CullingThe influence of human activities on the evolutionary trajectory of wildlife is widespread . Altered landscapes, climate change, invasive species, and direct harvest are shaping the genetic potential of species worldwide . In receC. latrans) expansion eastward (see C. lycaon) (C. rufus)   and red Seemingly limited to regions in and around Algonquin Provincial Park APP; , easternUnlike gray wolves in the west, eastern wolves readily hybridize with coyotes , and it C. lupus lycaon) that at the time was abundant across Ontario. Within the park, wolves have survived a long history of control efforts dating back to the park's establishment in 1893. Prior to the mid-1960s, wolves were actively poisoned, snared, and shot by park rangers in an effort to bolster game populations. Between 1909 and 1958, an average of 49 wolves per year (range 11–128) were killed in APP . Since Although wolf harvest in the first half of the 20th century presumably impacted the population size and altered the original genetic makeup of wolves within the park, the timing of the research culls in the mid-1960s is important because it occurred at a time when coyotes were becoming well established in the area. Prior to the 1960s, introgression from coyotes may have occurred, but was likely limited because the first coyote confirmed in southern Ontario was recorded in Thedford, Lambton County in 1919  and densAPP wolf samples used in this study were collected over 43 years in the different time periods: 1964–1965 (hereafter referred to as Historic Harvested [HH64–65]), 1987–1999 (hereafter referred to as Contemporary Harvested [CH87–99]), and 2002–2007 (hereafter referred to as Contemporary Protected [CP02–07]). Details regarding sample collection and DNA extractions for the CH87–99 samples can be found in n-lauroyl sarcosine, 10 mM CDTA , 0.1 M Tris-HCl, pH 8.0) and incubated at 37°C overnight (12–18 h). Then 50 µl of Proteinase K (600 mAU/mL) was added to each sample followed by incubation at 55°C overnight with rotation. Samples were then stored at 37°C up to 2 days to ensure complete digestion. Samples were extracted by standard phenol–chloroform methods adjusted for small volumes  to remove any foreign DNA and then rinsed with DNAase-free water . Teeth were crushed with a hammer to expose the inner vasculature and the dried blood from inside each tooth was placed in 400-µl 1× lysis buffer  were added to each reaction to account for 35 PCR cycles. Amplified product was visualized on an ethidium bromide stained agarose gel, and fluorescence was compared to a positive control with 500 pg of DNA in the reaction with the software Quantity One  to ensure that samples used in subsequent microsatellite reactions had at least 500 pg of DNA in each reaction and alleviate scoring errors due to allelic dropout  with sufficient target DNA, four Y chromosome microsatellite regions were amplified with primers MS34A, MS34B, MS41A, and MS41B  with 40 A 343- to 347-bp fragment of the mitochondrial DNA (mtDNA) control region was amplified from 2 ul of stock DNA with primers AB13279 and AB13280  under thMitochondrial DNA and Y microsatellite haplotypes were assigned based on previously published nomenclature  and compn = 17) were used in subsequent microsatellite analyses. Data included in microsatellite analyses include those generated here (HH64–65 and CH87–99) as well as previously published data from CP02–07, gray wolf–eastern wolf hybrids from northeastern Ontario (NEON), and eastern coyotes from southern Ontario along the Frontenac Axis (FRAX)  and NEON to FRAX at all six loci, then excluding cxx147 (five loci), and finally excluding locus cxx147 and cxx442  see . In the Fst) and tests of significant differences between populations based on 999 permutations. Jost's Dest  conducted Bayesian clustering analysis in Structure 2.2 (K) in Structure was determined by assessing a plot of the log probability of the data (Mean LnP(K)) and a plot of the second-order rate of change of the likelihood function (ΔK)  (which represent the posterior probability of membership to each cluster) over the 10 runs. PCA was conducted in the adegenet package  and these samples were excluded from the logistic regression analysis because assignment scores split across all populations can be an indication that the source population has not been sampled rather than representing influence from all populations.Measures of observed and expected heterozygosity, number of alleles, and private alleles were calculated in GenAlEx 6.3 , as weret's Dest  was also alleles . To asseture 2.2 , (3) useion (ΔK)  such tha package  of R . Sample sizes were consistent with observed data and Dest was calculated with a modified python script of SMOGD version 1.2.5 (Da) asThe coalescent simulations were generated with Serial SimCoal  within ADest  between on 1.2.5 . We wantDb) asand the relative change in differentiation from CH87–99 to CP02–07  gray wolves and North American-evolved (New World) species. The high ΔK values at K = 3 and K = 4 provide more subtle clustering information of more recently diverged groups. As shown in K = 4 is not biologically informative, thus K = 3 is suggested as the optimal number of clusters for this dataset.Analysis of the autosomal microsatellite data with Structure and PCA identified three main clusters in the dataset, with the three APP clusters having overlapping profiles  and 6, aP = 0.0397) but not from the mid-1960s to the 2000s  (P = 0.0236). Odds of sampling a gray wolf influenced animal were reduced by a factor of 0.21 in CP02–07 compared to HH64–65.Differences among the three Algonquin datasets were not readily obvious from these analyses. Results of the logistic regression, however, indicate a significant increase in the proportion of coyote-like animals in APP from the mid-1960s to the 1980/90s  . Odds ofDa) was 1.04, which was within the range, but greater than 93% (P = 0.06) of the coalescent simulations (Db) was –0.80 and lower than 95% (P = 0.05) of the simulations, suggesting the observed magnitude of gene flow from FRAX to APP was smaller than expected under constant migration across time periods.The observed relative change in population differentiation between HH64–65 and CH87–99 .The exact impacts and biological mechanisms of the mtDNA exchange are unclear, but a similar turnover of mtDNA haplotypes associated human-caused gray wolf extirpation followed by recolonization and subsequent dog introgression has been noted in Vancouver Island gray wolves . SimilarWe have shown that intensive eastern wolf culls may exacerbate hybridization with coyotes. These results may have implications for other closely related species that have been brought together by landscape changes and expansion of nonendemics. Wolves have been extirpated across most of their original range in North America with dramatic consequences for wolf viability and ecosystem health. For example, extirpation has led to widespread loss of genetic diversity within wolf populations thus reducing their adaptive evolutionary potential , and ecoC. lupus) is currently used to increase the size of ungulate populations in Alaska, USA (2) have increased over the past 22 years (Above all, our results demonstrate that intense localized harvesting of species thought to be numerous and widespread can have unexpected outcomes that threaten conservation of species and naturally functioning ecosystems. The advanced molecular genetic techniques now used for studying wildlife populations were unheard of in the 1960s and no one could have predicted the impacts that such an experimental design could have on a population. Although the research methods used in the 1960s would fail to meet current ethical guidelines, targeted culling is still common practice for managing wildlife under various scenarios . For exaska, USA , and in ska, USA  where bo22 years . Similar22 years . CoyotesOur results suggest the potential for ecological assumptions to be incomplete and that culling and other seemingly harmless, invasive methods, even when applied to abundant “pest” species, may have unexpected, lasting conservation implications. Whether for the purpose of game species management, protection of endemics, population size estimates, or collecting basic ecological knowledge, exploring nonlethal alternatives could minimize unanticipated impacts to animal populations and thus reduce the burden on wildlife managers. By following guidelines and principles of ecological ethics as outlined by a growing number of scientists , samplin