We find that, compared to water-treated control animals, fluoxetine-treated rats did not express higher levels of LTP and, in fact, exhibited reduced levels of potentiation at presumed intracortical A1 synapses. Bioactivity of fluoxetine was confirmed by a reduction of weight gain and fluid intake during the four-week treatment period. We conclude that chronic fluoxetine treatment fails to enhance LTP in the mature rodent thalamocortical auditory system, results that bring into question the notion that SSRIs act as general facilitators of synaptic plasticity in the mammalian forebrain.Several recent studies have provided evidence that chronic treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine can facilitate synaptic plasticity  in the adult central nervous system. Here, we assessed whether fluoxetine enhances long-term potentiation (LTP) in the thalamocortical auditory system of mature rats, a developmentally regulated form of plasticity that shows a characteristic decline during postnatal life. Adult rats were chronically treated with fluoxetine . Electrophysiological assessments were conducted using an anesthetized (urethane)  There currently is considerable interest in developing therapeutic strategies to enhance plasticity of the adult central nervous system (CNS). Physical exercise, diet, and various forms of environmental/cognitive enrichment have all been proposed to facilitate plasticity –4. In adInterestingly, recent reports have provided evidence for the notion that chronic treatment with the antidepressant and selective serotonin reuptake inhibitor (SSRI) fluoxetine can reduce the strength of GABAergic inhibition and promote plasticity of forebrain synapses. For example, chronic (4 weeks) fluoxetine administration restored ocular dominance shifts in the primary visual cortex (V1) of adult rats, a form of developmentally regulated plasticity that is significantly reduced in the mature brain . In addiThe notion that chronic SSRI treatment can exert a prominent, facilitating effect on plasticity has also been supported by investigations employing structural and neuroanatomical measures. Guirado et al.  noted thThe evidence summarized above indicates the potential of fluoxetine to affect plasticity of forebrain synapses. It is important to note, however, that some investigations have failed to detect beneficial effects (or noted adverse outcomes) of chronic fluoxetine treatment on plasticity or in animal models of several neurological diseases, some of which clearly involve deficient plasticity mechanisms  –20. ConsIn the present study, we assessed the effect of chronic fluoxetine treatment on LTP in the thalamocortical auditory system between the medial geniculate nucleus (MGN) and primary auditory cortex (A1) of adult rats. LTP in this projection system shows a sharp, age-dependent decline over postnatal life, with high levels of LTP present during the first 5-6 weeks of postnatal life, but only modest levels after postnatal day (PD) 100 . Here, wad libitum access to food and water. Individual housing was required in order to measure fluid intake for each animal. The colony room was maintained under a reversed 12 /12 hour dark/light cycle (lights on at 7 pm). Experimental procedures were performed in accordance with the published guidelines of the Canadian Council on Animal Care and approved by the Queen's University Animal Care Committee. All efforts were made in order to minimize animal suffering and the number of animals employed for these experiments.Experiments were conducted on adult, male Long-Evans rats . Rats were individually housed (cage dimensions 40 × 20 × 20 cm) with Mperiod = 4.5 and 4.7 weeks for fluoxetine and water rats, resp.), with fluid intake (every 48 hours) and body weight (every 7 days) recorded throughout the treatment period.All animals were allowed at least 1 week of acclimatization to the animal colony prior to the onset of fluoxetine treatment. Rats were randomly assigned to either the fluoxetine or water condition. The fluoxetine treatment regimen was the same as that described by Vetencourt et al. . FluoxetElectrophysiological assessments were carried out at the end of the treatment period (rat age of about 90–95 days) and followed previously established procedures , 22. Ratμm diameter Teflon-insulated stainless steel wire) was placed in A1. The final, ventral placement of both electrodes was optimized to yield the maximum amplitude of field postsynaptic potentials (fPSPs) in A1 in response to single pulse stimulation of the MGN.After anesthesia induction, rats were placed in a stereotaxic apparatus, the skull was exposed, and small holes were drilled over the MGN  and the ipsilateral A1 . Additional holes over the cerebellum and frontal cortex were used to secure reference and ground connections (jewelry screws), respectively. A stimulation electrode  was lowered into the MGN, and a recoding electrode , digitized , and stored for subsequent offline analysis. Stimulation (0.2 ms pulse duration) of the MGN was delivered by means of a stimulus isolation unit .For each rat, a 30 min period following the final electrode adjustments was given to allow for stabilization prior to the onset of data collection. Following stabilization, an input-output curve was generated by stimulating the MGN at successively increasing intensities . The intensity that elicited fPSPs of 50–60% of the maximal fPSP amplitude was chosen for the formal data collection.in vivo [Baseline fPSPs were recorded every 30 s until a 30 min period of stable baseline recordings was established. Subsequently, theta-burst stimulation (TBS) of the MGN was applied as trains of 10 bursts (delivered at 5 Hz), with each burst consisting of five pulses at 100 Hz. Stimulation trains were repeated every 10 s for a total of four trains. This induction protocol has previously been shown to elicit reliable LTP in the thalamocortical auditory system in vivo , 22. Aftμm) using a cryostat. The locations of all electrodes were examined using standard histological techniques and only animals with accurate placements were included in the analysis of the electrophysiology data.Immediately after the experiment, rats were perfused through the heart with 0.9% saline, followed by 10% formalin. Brains were extracted and immersed in formalin prior to sectioning . The fPSP amplitude was computed offline by Scope software . Values for each rat were averaged over 10 min intervals and these averages were normalized by dividing them by the averaged baseline (pre-TBS) amplitude of that animal. Data were statistically evaluated by repeated measures analysis of variance (ANOVA) and, if statistically appropriate, simple effects tests using the CLR ANOVA software package . The level of significance for statistical analyses was set at n = 18) or drinking water containing fluoxetine . During this period, weight gain was significantly reduced in rats given access to fluoxetine relative to control (water) animals , each rat was anesthetized with urethane to allow for the placement of a stimulating and recording electrode in the MGN and A1, respectively . Consistn = 11) and fluoxetine (n = 14) animals, respectively (P = 0.3).Initially, fPSPs elicited by single-pulse MGN stimulation were recorded for 30 min in order to establish a measure of baseline synaptic strength prior to LTP induction. Stimulation intensities used for the two groups of animals did not differ significantly, with MGN stimulation pulses of 0.48 ± 0.04 mA and 0.5 ± 0.02 mA for water (ectively . Furtherectively . The ampectively . It is nFollowing the completion of baseline recordings (30 min), two episodes of TBS were delivered to the MGN, each followed by 60 min of fPSP recordings. In water animals, TBS resulted in successful LTP induction, with the first TBS episode resulting in a potentiation of the first and second fPSP peak to 115% and 123% of baseline, respectively .In fluoxetine animals, TBS also resulted in LTP, but the amplitude of the first and second fPSP peak reached only 111% and 110% of baseline, respectively, following the first TBS episode . FurtherAs mentioned, the second fPSP peak in fluoxetine rats exhibits a baseline amplitude 23% higher than that seen in water animals. Even though this effect did not reach statistical significance, it might nevertheless indicate a minor enhancement of intracortical synaptic strength following chronic fluoxetine exposure. Such an enhancement may act to limit (occlude) further LTP induction by TBS delivery. In order to assess this possibility, we performed additional analysis by plotting and correlating baseline fPSP amplitude against levels of LTP for the second fPSP peak in all fluoxetine animals . HoweverWith the present experiments, we examined whether chronic fluoxetine treatment alters LTP of synapses in the mature A1 of adult rats. Several recent reports have provided support for the notion that chronic fluoxetine treatment leads to an enhancement of plasticity in the adult CNS –14, 25. in vivo typically consist of two distinct, negative peaks that correspond to current sinks associated with the successive activation of thalamocortical and intracortical A1 synapses, respectively [Previous work has shown that, in rats, fPSPs elicited by MGN stimulation ectively , 24, 26.ectively , 24. As The present data confirm that adult rats show modest levels of plasticity under the present experimental conditions, with thalamocortical and intracortical synapses expressing LTP of about 120% and 135% of baseline, respectively. Surprisingly, rats given chronic fluoxetine showed less LTP, with both thalamocortical and intracortical synapses expressing potentiation of only 113% of baseline, respectively, observations that are indicative of an inhibition of LTP induction mechanisms, especially for intracortical synapses in A1.The results summarized above regarding LTP in control animals are consistent with previous work, which has also shown that intracortical synapses in the adult A1 show higher LTP levels relative to thalamocortical synapses , 22, 24.We employed the same fluoxetine dosing regimen (0.2 mg of fluoxetine in 1 mL of drinking water) as that used in previous work showing a restoration of ocular dominance plasticity and enhancement of LTP in V1 slice preparations obtained from adult rats . In the While fluoxetine has been suggested to enhance plasticity of the mature CNS, empirical evidence assessing this contention has been inconsistent. Stewart and Reid  noted thin vitro approaches). It is also possible that some CNS regions  exhibit greater sensitivity to the potential, neurotrophic effects elicited by fluoxetine than areas such as the primary sensory fields of the neocortical mantle.The present experiments did not provide reliable evidence for an upregulation of A1 synaptic strength following chronic fluoxetine, with baseline fPSP amplitudes showing no significant differences between the two treatment groups. Previous work has found evidence for an enhancement of field potential strength in rats after fluoxetine administration in both the dentate gyrus and CA1 field , 30, evein vivo [Previous work has shown that local application of an NMDA receptor antagonist directly in A1 of rats blocks the induction of LTP elicited by MGN stimulation in vivo , 34. It in vivo , 35–37. in vivo , raisingin vivo , 30. FutIn recent years, there has been considerable excitement regarding the potential of various SSRIs to enhance CNS plasticity , 14, 39.


Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in men and the seventh in women and understanding the molecular mechanisms of HCC and establishing more effective therapies are critical and urgent issues. Our objective was to study the expression of ferroportin in hepatocellular carcinoma (HCC) tissue samples and the relationship between ferroportin expression and HCC characteristics.Sixty HCC tissues and their corresponding para-cancer liver tissues (PCLT) were obtained from sixty HCC patients who had undergone hepatectomy in the Second Xiangya Hospital of Central South University. Ten normal liver tissue samples were also obtained as a control. Immunohistochemistry (IHC) was performed to analyze the ferroportin expression in HCC, and the relationship between ferroportin expression and HCC clinical pathological characteristics also was analyzed. For the evaluation of IHC results, the comprehensive scoring criteria were met according to the staining intensity and the number of positive staining cells. Western blotting was performed to detect the expression level of ferroportin in HCC cell lines.P <0.05). Moreover, ferroportin expression was related to liver cancer cell de-differentiation, the severity degree in TNM staging, Edmondson-Steiner grading, intrahepatic metastasis and portal vein invasion. In addition, high expression of ferroportin was observed in normal human liver cell lines L02 and HL7702, whereas weak positive expression and even negative expression of ferroportin were observed in HCC cell lines FOCUS, MHCC-97H, HepG2 and SMMC-7721. Furthermore, among the four kinds of HCC cell lines, the expression level of ferroportin was the lowest in MHCC-97H cells.Ferroportin expression in HCC tissue was significantly lower compared to PCLT and normal liver tissue (Ferroportin expression level declines along with the progression of liver cancer, suggesting that the reduction of ferroportin may serve as an important marker for poor HCC prognosis and as a new therapeutic target. Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in men and the seventh in women, being one of the most lethal forms of malignancies with less than 10% survival rate ,3. Most Iron, an essential element for normal cell function, has been linked to tumor cell growth for a long history in experimental and clinical literatures ,7. High Ferroportin  is the only known mammalian export protein for nonheme iron -20. FerrThe role of ferroportin in cancer has not been extensively studied ,27. In 2Now that ferroportin has an important role in breast cancer, how about HCC? Ferroportin has a high expression level in liver and hepatocytes, and liver is the pivotal organ for iron metabolism -20,29. FAll the human tissue samples were obtained from the Second Xiangya Hospital of Central South University. We collected three kinds of samples: 1) For the HCC group we collected the HCC tissue samples from all HCC patients who had undergone hepatectomy in the Second Xiangya Hospital of Central South University during the years 2007 to 2011. These HCC patients had never received other therapies such as transcatheter arterial chemo-embolization, chemotherapy, or radiotherapy. All specimens had been confirmed by pathological diagnosis to be primary HCC. 2) For the para-cancer liver tissues (PCLT) group we took the PCLT that were located more than 2 cm from the tumor edge that had no necrosis from the HCC patients’ sample stated above. All these samples had gone through pathological examination and found to be free of cancerization and invasion. 3) For the normal liver tissue group we collected 10 normal liver tissue samples from patients without viral hepatitis history. All specimens were collected within half an hour of being detached from the body. For the use of these clinical materials for research purposes, prior consent from the patients and approval from the Ethics Committees of the Second Xiangya Hospital of Central South University were obtained. The specimens of the three groups above were tested by immunohistochemistry (ICH) to show the expression level of ferroportin.All 60 specimens from the HCC patients have undergone clinical and pathological diagnosis, and the HCC diagnostic criteria were met according to the criteria of World Health Organization. Tumor differentiation degree was classified by Edmondson-Steiner grading. Liver function was analyzed according to Child-Pugh score. Tumors were staged according to the sixth edition Tumor Node Metastasis (TNM) staging set by Union for International Cancer Control. The clinical data and pathological material were recorded. The basic clinical characteristics of HCC patients are shown in Table IHC was performed according to standard protocol. Paraffin tissue sections were deparaffinized in turpentine and rehydrated in a descending ethanol series according to standard protocols. Heat-induced antigen retrieval was performed in 0.01 mol/L citrate buffer at 100°C for 20 min. Antigen retrieval by enzyme digestion was performed in 0.1% trypsin at 37°C for 25 min. Sections were then blocked with serum for 30 min followed by incubation with rabbit anti-SLC40A1 antibody  for 1 to 2 hours at 37°C. After washing, the sections were incubated with horseradish peroxidase-goat anti-rabbit antibody at 37°C for 30 minutes. The peroxidase reaction was developed with 3, 3-diaminobenzidine (DAB) solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin, mounted in neutral gum and analyzed using a bright field microscope. A nasopharyngeal carcinoma pathological section was used as a negative control, and the primary antibody was substituted with PBS.For the evaluation of IHC results, the comprehensive scoring criteria were met according to the staining intensity and the number of positive staining cells: 1) the scoring criteria for staining intensity: 0, no staining; 1, weak staining; 2, modest staining; 3, strong staining; and 2) the proportion of tumor cell staining: 0, no positive tumor cells; 1, ≤ 25% positive tumor cells; 2, 25 to 50% positive tumor cells; 3, 51 to 75% positive tumor cells; 4, >75% positive tumor cells. The final score was calculated by adding the tumor staining area and the intensity score. A final score of 0 was treated as 0, negative expression; staining scores 1 to 3 were treated as 1, a weak positive expression; 4 and 5 were regarded as 5, an intermediate positive expression; and 6 and 7 were regarded as 7, a strong positive expression. Two pathologists scored all samples blindly without knowing clinical characteristics.FOCUS, HepG2 and SMMC-7721 are HCC cell lines, whereas MHCC-97H is a highly metastatic HCC cell line. L02 and HL7702 are normal human liver cell lines. All the cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum.Equal amounts of total extract protein (50 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted with polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skim milk at room temperature for 1 hour and washed, membranes were probed with rabbit-anti-SLC40A1  and mouse anti-β-actin  at 4°C overnight and then incubated with anti-mouse or anti-rabbit IgG antibodies conjugated with horseradish peroxidase. After final treatment with enhanced chemiluminescence assay (ECL) reagents , samples were exposed to X-ray film to detect and record relevant protein bands.t-test of independent samples were used to analyze comparisons of means. All the data were presented as mean ± S.D. The comparison of count data was analyzed by nonparametric test. Statistical difference was accepted at P <0.05.All statistical analyses were analyzed using SPSS 17.0 software . The ANOVA and In this study, IHC was used to evaluate the expression level and detect the location of ferroportin in HCC tissue sections (60 cases) and PCLT sections. The result demonstrated that ferroportin had two kinds of expression patterns in normal liver cells: 1) ferroportin showed obvious brownish black staining in the cytoplasm and cell membrane among most of the liver cells Figure .P <0.05). However, there was no significant difference in ferroportin positive expression rate and positive staining score between PCLT group and normal liver tissue (P >0.05).Ferroportin positive expression rate and positive staining score in the HCC group were significantly decreased compared to the PCLT group and the normal liver tissue group (P = 0.008). A similar relationship was also found between ferroportin expression and Edmondson-Steiner grading (P = 0.002). In addition, ferroportin expression was significantly lower in groups with intrahepatic metastasis and portal vein invasion than those without (P = 0.007). However, there was no significant relationship between ferroportin expression and alpha-fetoprotein(AFP) level (P = 0.18) , the lowest modest staining rate n = 1, 3.8%) and strong staining . However, in the I to II stage group, negative staining cases reduced greatly  and modest positive staining and strong positive staining increased to n = 5, 14.7% and n = 22, 64.7%, respectively. The expression level of ferroportin protein decreased with the increase of TNM .Given that there was a negative relationship between ferroportin expression and several HCC clinical characteristics, what is the situation of ferroportin expression in HCC cell lines? Can these HCC cell lines act as research tools for such kind of studies? To this end, we studied the expression level of ferroportin in HCC cell lines. We utilized immunoblotting to test ferroportin expression in four HCC cell lines  and two normal human liver cell lines (L02 and HL7702). The results demonstrated that high expression of ferroportin was observed in normal human liver cell lines L02 and HL7702, while weak positive expression and even negative expression of ferroportin was observed in HCC cell lines FOCUS, MHCC-97H, HepG2 and SMMC-7721 . Meanwhile, high ferroportin expression has been identified in a previous breast cancer study [Ferroportin is a transmembrane protein that transports iron from the inside of a cell to the outside of it and is a critical protein for the regulation of systemic iron homeostasis . In addier study . Thus, wet al. revealed that hepcidin served as an inhibitor for ferroportin, leading to the retention of iron within cells and a reduction in iron levels within the plasma [Recent research has suggested that ferroportin-mediated iron export is controlled by the hormone hepcidin, which binds ferroportin, causing its internalization and degradation . Moreovee plasma . Thus, tet al. have reported that ferroportin variant is associated with increased ferritin levels in African-Americans and may play a role in their propensity to develop iron overload [To our knowledge, mutations in the ferroportin gene can cause an autosomal dominant form of iron overload known as type IV hemochromatosis or ferroportin disease . The effoverload . In addioverload . Moreoveoverload . These fOur research results demonstrate that ferroportin expression in HCC tissue is significantly lower in comparison to PCLT and normal liver tissue, and that ferroportin expression is correlated with the clinical characteristics of HCC. All these results reveal that ferroportin expression level is decreased with the progression of liver cancer, suggesting that a reduction in ferroportin might be an important marker for poor HCC prognosis.in vitro study of the relationship between ferroportin and HCC as well as the underlying mechanisms. Will the growth rate of HCC cell lines change if we overexpress or knockdown ferroportin? What is the situation in vivo in an animal model? All in all, a better understanding of these might be helpful to the prognosis or therapy of HCC.However, additional investigation is needed to clarify the role of ferroportin in HCC and its crosstalk with other proteins. The ferroportin expression in the HCC cell lines that we have shown above may provide some useful information for the AFP: Alpha-fetoprotein; HCC: Hepatocellular carcinoma; PCLT: Para-cancer liver tissues; TNM: Tumor node metastasis.The authors declare that they have no competing interests.QW and JZ participated in the design of this study, and they both performed the statistical analysis. DWZ carried out the study, QWW collected important background information, and drafted the manuscript. QW and JSH conceived of this study, and participated in the design and helped to draft the manuscript. All authors read and approved the final manuscript.


Schizophrenia is a chronic and often debilitating illness which affects about 1% of the world population. Some reagents have been used to simulate schizophrenic disorders in laboratory animals, such as amphetamine and ketamine. Previous studies have suggested that reactive oxygen species (ROS) production, reduced levels of ATP, mitochondrial dysfunction and apoptosis are involved in the pathophysiology and etiology of schizophrenia. In this study we divided Wistar rats in to 2 groups; control group received normal saline and test group received ketamine 30 mg/Kg daily for five consecutive days. Then, locomotor activity including side to side head rocking and arcing of neck, proved schizophrenia in the test group rats. Rats in both control and test groups were then decapitated and brain mitochondria were isolated. Our results showed increased ROS formation, mitochondrial membrane potential collapse, mitochondrial swelling and cytochrome c release in mitochondria of schizophrenic test group. Our findings suggested that mitochondrial ROS formation and apoptosis signaling are likely involved in cellular pathology of Schizophrenia. To our knowledge this is the first report that provides a mechanistic justification between mitochondrial events and neuodegeneration in the Schizophrenia.  Schizophrenia is a mental disorder characterized by a breakdown of thought processes and by a deficit of typical emotional responses . PrevaleAlthough cellular and molecular pathology and etiology of schizophrenia have not yet been elucidated, but devastated mitochondrial function and as a consequence impaired cellular energy state, is an attractive hypothesis for describing the pathophysiology of schizophrenia. MitochoThere are a number of scrambles which want to explain the link between altered brain function and schizophrenia . One of In this study we used ketamine for simulation of schizophrenic disorder in Wistar rat, and then we isolated mitochondria from brain of schizophrenic rat to figure out probable mitochondrial toxic mechanisms such as collapse of mitochondrial membrane potential, mitochondrial swelling, the raise of active oxygen radicals and finally release of cytochrome c which is the start point of cell death signaling.AnimalsMale Wistar rats (250-300 g) were housed in an air-conditioned room with controlled temperature of 25 ± 2 °C and maintained on a 12:12 h light cycle with free access to food and water. All experimental procedures were conducted according to the ethical standards and protocols approved by the Animal Experimentation Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran. All efforts were made to minimize the number of animals and their suffering.Drugsi.p) injected at a volume of 1 mL/Kg body weight. All solutions were freshly prepared before injection. Ketamine was purchased from Sigma-Aldrich Co  and was dissolved in physiological saline. The drugs were intraperitoneally  at 4 °C, except for the mitochondria used to assess ROS production, MMP and swelling, which were suspended in respiration buffer , MMP assay buffer  and swelling buffer . Protein concentrations were determined through the Coomassie blue protein-binding method as explained by Bradford, 1976 (26). The isolation of mitochondria was confirmed by the measurement of succinate dehydrogenase (27) .Mitochondria were prepared freshly for each experiment and used within 4 h of isolation and all steps were strictly operated on ice to guarantee the isolation of high-quality mitochondrial preparation.Determination of mitochondrial ROS levelThe mitochondrial ROS measurement was performed using the fluorescent probe DCFH-DA. Briefly, isolated brain mitochondria were placed in respiration buffer containing 0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate . FollowiDetermination of the MMPMitochondrial uptake of the cationic fluorescent dye, rhodamine 123, has been used for the estimation of mitochondrial membrane potential. The mitochondrial fractions (0.5 mg protein/mL) were incubated with 10 μM of rhodamine 123 in MMP assay buffer . The fluorescence was monitored using Shimadzu RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively .Determination of mitochondrial swellingAnalysis of mitochondrial swelling after the isolated mitochondria (0.5 mg protein/mL) was estimated through changes in light scattering as monitored spectrophotometrically at 540 nm (30 °C) as described . BrieflyCytochrome c release assayThe concentration of cytochrome c was determined through using the Quantikine Rat/Mouse Cytochrome c Immunoassay kit provided by R and D Systems, Inc. . Briefly, a monoclonal antibody specific for rat/mouse cytochrome c was pre-coated onto the microplate. Seventy-five μL of conjugate  and 50 μL of control and test group were added to each well of the microplate. One microgram of protein from each supernatant fraction was added to the sample wells. All of the standards, controls and test were added to two wells of the microplate. After 2 h of incubation, the substrate solution (100 μL) was added to each well and incubated for 30 min. After 100 μL of the stop solution was added to each well; the optical density of each well was determined through the aforementioned microplate spectrophotometer set to 450 nm.Statistical analysisResults are presented as mean ± SD. Assays were performed in triplicate and the mean was used for statistical analysis. Statistical significance was determined using the student t-test or one-way ANOVA test, followed by the post-hoc Tukey test when appropriate. Statistical significance was set at P < 0.05.The characteristic behavioral responses including side-to-side head rocking and arching of the neck were evaluated. Ketamine treated rats showed significant increase (P < 0.001) in both side-to-side head rocking and arching of the neck compared to control rats .P < 0.05) increased in isolated brain mitochondria obtained from ketamine (30 mg/Kg i.p. × 5 days) treated rats compared to control rats. The ROS formation in two groups was measured in the time intervals  following isolation of rat brain mitochondria in both groups.As shown in Moreover, we determined the mitochondrial swelling, an indicator of mitochondrial membrane permeability (MPT), by recording the decrease of absorbance of isolated mitochondria obtained from ketamine treated rats at 530 nm (A540). Our results showed significant mitochondrial swelling (p < 0.05) in the mitochondria isolated from brain of ketamine treated test group compared with control group . The mitMitochondrial membrane potential is a highly sensitive indicator of the mitochondrial inner membrane condition was measured by Rhodamine 123 redistribution. As shown in Cytochrome c release, the endpoint of mitochondrial toxicity, was significantly (p < 0.05) increased in the mitochondria isolated from brain of ketamine treated test group compared with control group .Schizophrenia is a chronic and often debilitating illness which affects about 1% of the world population. In addition to severely disrupting the life of the patient and their family, schizophrenia incurs a great cost to society in terms of lost productivity and treatment-related expenses. Although the etiology remains unknown, converging lines of evidence, including reduced gray matter volume, reduced synaptic markers and reduced neuropil, suggest that disrupted cortical synaptic circuitry is a central deficit in schizophrenia . The undSome models have been used to simulate schizophrenia, such as amphetamine and ketamine. In this study we induced schizophrenia by ketamine and used (30 mg/Kg × 5 consecutive days) ketamine for stimulation of schizophernia in the rats. Then we assessed the locomotor activities including side to side head rocking and arcing of neck in two groups of ketamine treated and unexposed control rats. Our results showed that the locomotor activities in the ketamine treated group were significantly higher than control group  and cytochrome c release. Previous studies showed that MMP represents the integrity of the mitochondrial membrane and its metabolic activity as a key indicator of mitochondrial functionality . FurtherAltered regulation of the apoptotic cascade can potentially reduce neuronal and glial viability at various stages of neural development and contribute to the volumetric and functional brain deficits observed in schizophrenia .In parti


Agelaius phoeniceus) show a mosaic phenotypic distribution, with a “bicolored” form occurring disjunctly in California and Mexico. We analyzed the relationships among these bicolored populations and neighboring typical populations, using ∼600 bp of mitochondrial DNA sequence data and 10 nuclear short tandem repeat loci. We find that bicolored populations, although separated by ∼3000 km, are genetically more similar to one other than they are to typical populations separated by ∼400 km. We also find evidence of ongoing gene flow among populations, including some evidence of asymmetric gene flow. We conclude that the current distribution of bicolored forms represents incomplete speciation, where recent asymmetric hybridization with typical A. phoeniceus is dividing the range of a formerly widespread bicolored form. This hypothesis predicts that bicolored forms may suffer extinction by hybridization. Future work will use fine-scaled geographical sampling and nuclear sequence data to test for hybrid origins of currently typical populations and to more precisely quantify the directionality of gene flow.Geographic distributions of genetic and phenotypic characters can illuminate historical evolutionary processes. In particular, mosaic distributions of phenotypically similar populations can arise from parallel evolution or from irregular patterns of dispersal and colonization by divergent forms. Two phenotypically divergent forms of the red-winged blackbird ( Speciation appears to take place most often in allopatry , behavioral characters , or sexual competitiveness . Such asymmetries imply that differentiation is not always a neutral process and that differences acquired in allopatry – along with the selective forces that favor them – have important implications for the success of new lineages. Moreover, these asymmetries may be an underappreciated factor in community assembly and stability  were synonymized with A. phoeniceus in 1931  and central Mexico  indicates that admixture of bicolored and typical blackbirds is much more widespread than reported in the 1960s . Cycling conditions consisted of 30 cycles of 20 s denaturation at 95°C, 20 s annealing at 55°C, and 45 s extension at 72°C, followed by a 3 min extension at 72°C. For toe pad extractions, reactions were identical to those above, but used 35 cycles instead of 30, and the time for each step was doubled . Most PCR products from toe pads were then reamplified with 25 additional cycles prior to sequencing, in order to increase concentration of PCR product. Because we were not always able to amplify CR in one fragment from toe pad extractions, we designed the following internal primers to amplify and sequence CR in two fragments: BBCR1_intF (5′-CACCTTTGAATTCCCCTAGTCT-3′) and BBCR1_intR (5′-GCTTGGGTGYTCYTGAAGGCT-3′).DNA was extracted with DNEasy kits  according to the manufacturer's protocols. For toe pad samples, we added 20 μmol of dithiothreitol (DTT) in aqueous solution to the digestion stage to aid in keratin proteolysis and concentrated final elutions by ethanol precipitation and resuspension of DNA in 1/5 the normal volume of buffer. We sequenced 595 bp of the mitochondrial DNA control region (CR) from each sample. The sequenced region is highly variable and information dense .All products were purified with Exonuclease and shrimp alkaline phosphatase  and sequenced using ABI BigDye, version 3 chemistry, and an ABI 3730XL capillary sequencer . All fragments were sequenced in both directions. Reads were assembled, trimmed, and edited in Sequencher, version 4.1 . Sequences were aligned by hand; alignment of A. phoeniceus, which were previously tested for optimal annealing, consistent amplification, homology, and variability in both bicolored and typical populations . For toe pad extractions, primer quantities were adjusted to increase the quantity of fluorescently labeled PCR product. These reactions were as above, but with 1.25 pmol forward primer, 5 pmol reverse primer, and 5 pmol dye-labeled M13 primer. Cycling consisted of 25 cycles of 15 s denaturation at 95°C, 15 s annealing at primer-specific temperatures, and 20 s extension at 72°C, followed by 10 similar cycles with 53°C annealing temperature, and finally a 7 min extension at 72°C.We genotyped each individual at five short tandem repeat (STR or microsatellite) loci  developed specifically for GeneMarker, version 1.91 . Raw size data were binned using the method of Idury and Cardon  and reference fragments to assess variation across runs and dyes. Size scores were estimated by the analysis of electropherograms in d Cardon  implemenBEAST, version 1.7.2 . We then assigned individuals to clusters using discriminant functions. We determined the appropriate number of discriminant functions to retain by estimating the optimal α score, using randomization of alleles to ensure that models were not overfit. Finally, we plotted individuals on the discriminant function axes in order to visualize variation within and among the sampled populations.In addition to population-level analyses, we clustered individuals using two complementary analyses of the STR data. First, we inferred genetic clusters using discriminant analysis of principal components  values, we estimated the posterior probability for each value of k and selected the value of k with the highest probability for subsequent analyses. We then performed 100 runs for this value of k, aggregated runs in clumpp  to nearly 0.16 (between Lerma and Veracruz), and similar variability was shown by other loci and measures of differentiation  to the exclusion of the two typical populations . The concordance between the DAPC and Structure results is notable, as it indicates that the inferred number of genetic clusters is insensitive to the different assumptions of these two methods. In addition, the assignment of individuals to clusters was strongly correlated between the two methods .As with the DAPC analysis, s 2 Fig. , with a s 2 Fig. . In runsP < 0.01). This model implies dispersal from California to Mexico, with little or no dispersal in the other direction (Table P = 0.15), in this case implying higher dispersal rates from Shasta Valley to Black Point than the reverse.We found strong evidence of gene flow between populations. First, in all cases, the model with minimum AIC included migration Table . In addion Table . The secm1→2 + m2→1]/2) between Black Point and Lerma estimated by coalescent analyses of all the data were higher than between any other pair, although this difference is not statistically significant , to abrupt transitions . The presence of multiple abrupt transitions between phenotypes yields a “mosaic” pattern, where isolated populations may all be different from one another, or where some populations (adjacent or otherwise) share phenotypic characteristics. Mosaic patterns of variation, with geographically disjunct but phenotypically similar forms – as seen in the red-winged blackbird – are particularly interesting, as there are relatively few evolutionary processes that can produce them, and these processes are of intrinsic interest. On the one hand, adaptation in a heterogeneous landscape can drive population differentiation, and similar phenotypes may arise if disjunct regions share similar selective environments . Alternatively, differentiation in allopatry, followed by partial displacement of one postisolation form by another, can yield similar disjunct patterns. Examples of both these patterns have been reported previously, but are relatively rare.Gasterosteus aculeatus) has revealed repeated, independent evolution of multiple locally adapted freshwater forms from an oceanic ancestor. These disjunct forms show consistent phenotypic changes related to locomotion and feeding in different habitats (Schluter and McPhail Chaetodipus intermedius) has evolved dark coat coloration on multiple disjunct lava flows in the desert southwest. Although quite similar in pelage, these populations are not distinct from one another or surrounding populations based on mtDNA  suggests that this pattern is an historical species difference rather than local adaptation, most likely due to contraction of P. shermani and expansion of P. teyahalee distributions since the last deglaciation , which are behaviorally dominant to red wings and currently sympatric with them in California  rather than local selection. Notably, there is nothing apparent in the environments of central Mexico and California that might select for the observed plumage differences, particularly parallel changes in both male and female plumage. We are currently conducting analyses of environmental similarities between these regions using niche modeling  as one of a growing list of examples of asymmetric hybridization and suggest that the biological determinants of this phenomenon in blackbirds merit additional study.Cases of asymmetric hybridization between closely related but incompletely isolated forms offer insight into the processes influencing species differentiation at its earliest stages. Recognition and analysis of such cases contributes significantly to the study of speciation. In particular, the fact of asymmetric range expansion reveals the existence of biologically meaningful differentiation – be it demographic, behavioral, or otherwise – between hybridizing forms. Comparative analysis of multiple lineages showing such characteristics may reveal generalities regarding the selective factors that drive such differences  or the lack of such . Here, we offer red-winged blackbirds (


Several studies have reported that pancreatic ductal preservation greatly improved the islet yield and function after cold storage. However, these studies were devoid of appropriate controls, such as vascular perfusion, which is routinely performed to preserve organs in the clinical setting. In this study, we created a vascular perfusion model using inbred rats, and investigated the effect of ductal injection on the islet yield and function after cold storage. Rat pancreases after 10 h cold ischemia were classified as follows: without ductal/vascular perfusion; with ductal injection; with vascular perfusion; and with ductal/vascular perfusion. The islet yield, function, viability, release of inflammatory mediators, and pathological changes in the exocrine tissues were assessed in the Hanks' Balanced Salt Solution (HBSS) model. The islet yield was also assesed by introducing University of Wisconsin Solution (UWS) and Histidine-Tryptophan-Ketoglutarate solution (HTK), which are the standard clinical preservation solutions. In the HBSS model, ductal injection and vascular perfusion significantly improved the islet yield compared with the control group. However, ductal injection showed no additional effects on the islet yield, function, viability and suppressing the release of inflammatory mediators when vascular perfusion was performed. Although ductal injection significantly decreased the apoptosis of exocrine cells, no beneficial effect on vacuolation was observed. In contrast, vascular perfusion significantly suppressed vacuolation in the exocrine tissues. Likewise, in the UWS and HTK model, ductal injection and vascular perfusion improved the islet yield compared with the control group. Nevertheless, the combination group showed no additional effects. These data suggest that ductal injection has no additional effect on islet yield and function after cold storage in a vascular perfusion model. We propose that ductal injection can be an effective and simple alternative for vascular perfusion prior to pancreas harvest, but is not necessary in most cases, since vascular perfusion is routinely performed. Islet transplantation is a promising strategy for curing type 1 diabetic patients. Although Shapiro et al. greatly improved the clinical outcome Although several factors may contribute to the failure of islet isolation, ischemic stress during pancreatic harvesting and preservation has been known to deteriorate islet recovery and function It has been reported that a pancreatic ductal preservation method greatly improved both the islet yield and function after cold ischemia per se may have a beneficial influence on the islet yield and function. Therefore, to properly elucidate the effects of the ductal preservation method, experimental models with vascular perfusion need to be examined.Of particular note, however, the previous studies on the effects of ductal injection were devoid of appropriate control groups, namely a vascular perfusion group. In the current clinical setting, vascular perfusion using cold preservation solutions is routinely performed to preserve organs at the time of harvest from cadaveric donors. It seems likely that vascular perfusion In the present study, we hypothesized that a ductal perfusion does not have an additional effect on protecting the pancreas from the ischemic stress when vascular perfusion is simultaneously performed. At the first step, we investigated the effects of vascular perfusion and/or ductal injection on the islet yield and function after a 10 h cold ischemia time (CIT) using the Hanks' Balanced Salt Solution (HBSS) in a vascular perfusion model of inbred rats, which were expected to show minimal individual differences. We also examined whether ductal injection could regulate the release of inflammatory mediators from the isolated islets. Futhermore in order to mimic clinical situation, University of Wisconsin Solution (UWS) and Histidine-Tryptophan-Ketoglutarate solution (HTK) were applied at the seconed step. In the UWS and HTK model, we assesed the effects of vascular perfusion and/or ductal injection on the islet yield after a 30 h and 10 h CIT.In the HBSS model , the isl14/mol s−1, ductal injection 3.12±0.20×1014/mol s−1, vascular perfusion 3.58±0.22×1014/mol s−1, combination 4.28±0.52×1014/mol s−1, n = 6, respectively ; ATP/DNA ratio: control 40.1±1.6 pmol/µg (n = 6), ductal injection 40.4±1.6 pmol/µg (n = 7), vascular perfusion 40.3±1.0 pmol/µg (n = 7), combination 42.8±2.0 pmol/µg (n = 6)). For all viability assays performed, no significant differences were detected among the groups.A static glucose stimulation (SGS) test  was perfTo investigate the effects of ductal injection and/or vascular perfusion on the energy status of exocrine tissues, the ATP/DNA ratio of exocrine tissues was examined . The ATP2) and combination groups (74±31/mm2) compared with that of the ductal group (415±31/mm2) . The vacuolation density of the combination group was also significantly lower than that of the control group (349±35/mm2) (P<0.01). As summarized in The DNA fragmentation of islets and exocrine tissues was evaluated by the TUNEL (TdT-mediated dUTP nick end labeling assay) to assess the extent of apoptosis. Representative examples are shown in Isolated islets subjected to ischemic stress, especially warm ischemia in vitro assays after cold storage when vascular perfusion was performed. Of particular note, the same tendency was observed when the potent preservation solution was applied.Several studies have reported that pancreatic ductal preservation greatly improved both the islet yield and function after cold ischemic storage Corroborating our hypothesis, the TUNEL staining in the present study revealed that cold ischemia substantially damaged the exocrine tissues, rather than the pancreatic islet cells. Moreover, it was also shown that ductal injection could significantly protect the exocrine tissues from the apoptotic changes. Unexpectedly, vascular perfusion had only a marginal effect on inhibiting the apoptotic changes of the exocrine tissues. However, vascular perfusion, unlike ductal injection, efficiently suppressed vacuolation in the exocrine tissues after cold preservation. In the present study, frequent vacuolation was observed in the pancreatic tissues after cold storage, while only a few apoptotic cells were detected. Considering the limited apoptotic changes, it may be speculated that vacuolation could be a useful marker to detect the early phase of ischemic damage. This finding is consistent with a previous report by Monbaliu et al In the present study, we also observed several necrotic areas in the pancreatic acini, but only in the ductal injection and combination groups. Given that no necrotic areas were detected in the control group, these changes seem to have been induced by the ductal injection. However, the previous studies reported that both pancreatic duct cells and the exocrine tissues were preserved when ductal injection was performed prior to pancreas preservation Another possible explanation for the discrepancy between the present study and the previous studies is the animal model used. For islet isolation, collagenase injection through the pancreatic duct and distension of the pancreatic tissues are definitely critical and essential for pancreas digestion. In small animal models, collagenase delivery seems to be of less importance than in humans, even after cold storage. In large animals, it may have a stronger impact on the islet yield, since the ductal components and structures are complicated. Further investigations using both ductal and vascular perfusion models in a large animal setting are required to optimize the ductal injection procedure and to determine whether it can improve the islet viability or function.In support of the previous findings It was previously reported that isolated islets subjected to cold ischemic stress produce inflammatory cytokines In conclusion, the present data suggest that ductal injection has no additional effect on the islet yield and function after cold storage when vascular perfusion is performed in rat model. However, we propose that ductal injection can be used as an effective and simple alternative for vascular perfusion prior to pancreas harvesting. Further investigations using both ductal and vascular perfusion models in a large animal setting are required.All animals in this study were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Instituted of Health per se. UWS and HTK are potent preservation solutions and have commonly been used for clinical islet transplantation. The donor rats were anesthetized using isoflurane  to minimize suffering. After injection of 100 IU of heparin, the common bile duct was cannulated using a 24 G SURFLO I.V. catheter  and fixed with surgical silk. The pancreas was perfused via cannulation of the abdominal aorta using a 24 G SURFLO I.V.  and then it was flushed with 50 ml of cold HBSS at a pressure of 100 cm H20 or cold UWS at 5 ml/min using a syringe pump  after clamping the abdominal aorta above the celiac axis. Immediately after starting perfusion, the intrathoracic inferior vena cava (IVC) was dissected for outflow. At the aorta clamp, crushed ice was placed into the abdominal space to reduce the organ temperature during perfusion. In the groups without vascular perfusion, crushed ice was placed similarly, and the rat was left for 7 min in the HBSS model or 10 min in the UWS and HTK model (the same duration as the perfusion time). After vascular perfusion, the pancreas, along with the spleen, duodenum and stomach, was removed as en bloc, and ductal injection was performed via slow injection of 0.5 ml of HBSS, UWS, or HTK through a common bile duct catheter. The distal bile duct was not clamped during a ductal perfusion to avoid excess pressure. And also, the catheter remained open during the entire preservation period. The pancreas was preserved in HBSS, UWS, or HTK at 4°C. In the present study, the CIT was defined as the time from clamping the aorta to the initiation of islet isolation. Pancreases were classified into 5 groups. In the fresh group, the pancreases were immediately isolated. The pancreases subjected to 10 h CIT in the HBSS and HTK model or 30 h CIT in the UWS model were classified as follows: with neither ductal injection nor vascular perfusion (control group); with ductal injection ; with vascular perfusion (vascular group); with ductal injection and vascular perfusion (combination group). In the warm ischemia time (WIT) model, the crushed ice was removed at the end of vascular perfusion, and the rat was left for 30 min. After that, the pancreas was procured similarly to the CIT model and preserved for 10 h in 4°C HBSS.In the present study, HBSS, UWS , and HTK  were used for vascular perfusion, ductal injection, and pancreas preservation. HBSS, which is a simple buffer solution, was employed to focus on the comparison between the ductal injection and vascular perfusion procedures The pancreas was distended using 1 mg/ml of a collagenase  solution in 10 ml HBSS through a common bile duct catheter. After the spleen, duodenum and stomach were removed, the pancreas was digested . Thereafter, density gradient centrifugation was performed as previously reported in vitro test for islet function, as descrived previously A SGS test was performed on the isolated islets as an The ATP/DNA ratios of 30–40 islets in each group and of the exocrine tissues were measured after cold preservation. Forty exocrine tissue blocks (200 µm in diameter) were selected for each assay. The ApoGlow™ kit  was used for the ATP measurements, as described previously 2 and humidified air for 72 h. After incubation, the supernatants were collected and frozen at −80°C until use. The islets in the wells were collected to measure the DNA content as previously described After isolation with cold and warm ischemia, 50 islets were collected from each ischemic group and placed into 48 well dishes  with 250 µl of RPMI 1640 containing 5.5 mmol/L glucose and 10% fetal bovine serum, at 37°C in COin situ was assessed by the Apoptosis Detection Kit . Slides (six sections per pancreas and three pancreases per group) were subjected to the TUNEL assay, and evaluated with regard to islets and exocrine tissues. In each section, the percentage of positive islets  and the density of positive cells in the exocrine tissues ) were determined. The vacuolation and the necrotic area in the exocrine tissues were also examined. Slides stained with hematoxylin and eosin (H&E) (two sections per pancreas and three pancreases per group) were examined. The density of vacuolation ) and the necrotic area ) in 10 random fields of each sample were calculated at 200-fold magnification.Pancreases from the ischemic groups were placed into 4% paraformaldehyde and embedded in paraffin wax. DNA fragmentation P value<0.05 was considered to be significant.All results are reported as the means ± standard error (SE). A one-way ANOVA followed by the Tukey test was used for the statistical evaluations. A


Clupea harengus), often show a low degree of differentiation over large geographical regions. Despite strong environmental gradients  in the Baltic Sea, population genetic studies have shown little genetic differentiation among herring in this area, but some evidence for environmentally-induced selection has been uncovered. The mitochondrial genome is a likely target for selection in this system due to its functional role in metabolism.Marine fish, such as the Atlantic herring (ST = 0.036). There was evidence for significant positive selection, particularly in the ND2, ND4 and ND5 genes, and amino acids under significant selection in these genes explained some of the clade formation. Despite uncovering evidence for selection, correlations between genetic diversity or differentiation with environmental factors  were weak.We sequenced whole mitochondrial genomes for herring from throughout the Baltic region (n=98) in order to investigate evidence for geographical structuring, selection, and associations between genetic and environmental variation. Three well-supported clades that predate the formation of the Baltic Sea were identified, but geographic structuring of this variation was weak (ΦThe results indicate that most of the current mtDNA diversity in herring predates the formation of the Baltic Sea, and that little structuring has evolved since. Thus, fisheries management units in this region cannot be determined on the basis of mtDNA variability. Preliminary evidence for selection underlying clade formation indicates that the NADH complex may be useful for examining adaptation and population structuring at a broader geographical scale. Marine fish often show weak genetic divergence over large geographical areas ,2. This th of nuclear genes; but see [Mitochondrial DNA (mtDNA) is a potentially useful source of informative markers for investigating population structuring and adaptation in marine species due to its low effective population size (1/4but see ) and docbut see ,12. Mitobut see  and funcbut see . Furtherbut see . Recombibut see .http://www.mitomap.org), to thermal adaptation [Parus spp.), based on the fact that certain populations consistently had a single non-synonymous substitution in the ND2 gene [Theragra chalcogramma) have been shown to have more mitochondrial diversity than expected under a model of mutation and genetic drift, indicating that selection is occurring along a North–south gradient in two separate sampling regimes on either side of the North Pacific [Several studies have detected specific mutations and regions of the mitochondrial genome that appear to be under positive selection. Mutations in mitochondrial genes have been linked to a large number of human diseases ; particD2 gene . In addiPacific .et al. [Mitochondrial DNA may be a particularly useful marker for marine species, as they are likely to experience strong selective pressures towards high aerobic capacity, and those with wide distribution ranges may also need to adapt to environmental clines . Broaderet al.  found evet al. . Althouget al. ,26.Clupea harengus) in the Baltic Sea provide an ideal case study to investigate mitochondrial genome variation and the possibility of selection, as this geographical region is characterised by strong environmental gradients and local adaptation to this variation may be expected. The Baltic Sea region, including the Skagerrak and Kattegat seas that connect the Baltic Sea to the North Sea, is a post-glacial environment, which has existed only for approximately the past 8,000 years [http://www.itameriportaali.fi/en/itamerinyt/en_GB/itamerinyt/). The Skagerrak and Kattegat seas mark the region where the environment changes from being strictly marine (North Sea) to brackish .The Atlantic herring  of whole mitochondrial genome sequences of herring from 16 localities covering the entire Baltic Sea, and an additional sample from the Barents Sea. First, is there any evidence for geographical structuring of genomic variation? Second, is there any evidence for positive selection acting on the mitochondrial genome? Third, is there any evidence that the genomic variation is associated with local variation in sea temperatures and salinity? We conclude by discussing the implications of our findings for the management of herring fisheries in the Baltic Sea.Sampling was designed to cover the whole Baltic Sea, with an additional sample from the Barents Sea Figure . HerringClupea harengus sequences as a template . PCR mixes for all primer pairs consisted of 5μl Qiagen Taq PCR Master Mix , 3μl H2O, 0.5μl of each primer at 10pmol, and 1μl DNA. The PCR program used for all primer pairs was: denaturation at 95°C for 15 min, followed by 30 cycles of 95°C for 30s, 60°C for 1 min 30s, 72°C for 1 min, followed by a final elongation step at 60°C for 10 min. The resulting products were run on 1% agarose gels to check for amplification, cleaned using Exonuclease I and FastAP , and Sanger sequencing was outsourced to Beckman Coulter Genomics (UK) and the Institute for Molecular Medicine (Finland). Sequences were checked manually, assembled, aligned, and annotated in Geneious Pro 5.4 [C. harengus mitochondrial genome (GenBank accession number AP009133). Note that the mtDNA genome has several overlapping genes , and one gene (ND6) is encoded on the complementary strand. Overlapping nucleotide sections were duplicated so that each gene sequence was fully represented in the individual gene and concatenated gene datasets, and the reverse complement of the ND6 nucleotide sequence was used in order to infer the correct amino acid sequence.The mitochondrial genome was amplified in 22 overlapping segments, using 22 novel primer pairs  whole genome (nucleotide sequence), (ii) each of the 13 individual genes and the control region as separate datasets, (iii) all genes concatenated .Clupea pallasii (GenBank accession number NC009578) was used as an outgroup to root both types of tree. A neighbour-joining tree was produced from the same dataset using between sampling site mean nucleotide distances (proportion of nucleotides at which two sampling sites are different) in MEGA v.5.05 [(i) Whole genome - The most suitable model of nucleotide evolution for the whole genome sequences was chosen using jModelTest 0.1.1 ,41 This  v.5.05 . In orde v.5.05 .Clupea pallasii (GenBank accession number NC009578) was used as an outgroup to root all trees.(ii) Individual genes and control region – Datasets for each of the 13 individual coding genes, and for the control region, were run through jModelTest 0.1.1 ,41. ThesClupea pallasii (GenBank accession number NC009578) was used as an outgroup to root the tree. An additional maximum likelihood tree was produced using amino-acid data for all genes concatenated with 500 bootstrap replicates. A neighbour-joining tree was produced with the same dataset, using between sampling site mean amino acid distances (proportion of amino acids at which two sampling sites differ) in MEGA v.5.05 [(iii) All genes concatenated - Trees were also created using the dataset of all coding genes combined. A Bayesian tree was created in MrBayes  partitio v.5.05 . Also, a v.5.05  for the C. harengus, and its intraspecific subclades was performed using a Bayesian relaxed clock method implemented in BEAST v.1.6 [C. pallasii GenBank accession numbers NC009578, AP009134; Sprattus sprattus AP009234, NC009593). Two nodes were used for temporal calibration based upon the phylogenetic relationships among 25 Clupeiforme mtDNA genomes [Sprattus and Clupea, thus a uniform prior between 60 and 10 millions of years ago (Mya) was chosen, with an initial value of 30 Mya. The TMRCA between C. harengus and C. pallasii was defined as a truncated normal distribution having a mean of 4 Mya, a standard deviation of 1 My, and an upper and lower limit of 3 and 5 Mya, respectively, based on previous estimates for the age of this node [6 iterations each, with a burn-in of 106. Samples were taken every 1000 iterations and operators were optimized automatically. Results of replicate runs, convergence, asympotic performance and effective sample size (ESS) of posterior parameters  were assessed using Tracer v1.5 [Estimating the Time to Most Recent Common Ancestry (TMRCA) of T v.1.6 . The allT v.1.6 ; C. pallgenomes . Nothingis node . The anaer v1.5 .ST estimator used was ΦST, a direct analogue of Wright's FST for nucleotide sequence divergence. Again, we applied the sequential Bonferroni correction, and a false discovery rate of 0.05. Arlequin was also used to calculate pairwise ΦST for the whole genome and any genes that indicated significant population structure.Analysis of Molecular Variance (AMOVA) was performed for the whole genome nucleotide dataset, for each individual coding gene, and for the control region, using Arlequin version 3.11  with 10,Isolation by distance tests to compare genetic and geographic distances were performed using Mantel’s tests (Pearson method) in R , using 1Selection is often assessed by looking at the ratio of nonsynonymous (dN) to synonymous mutations (dS), where a ratio of dN:dS > 1 is taken to indicate positive selection . Howeverhttp://ocean.ices.dk/Helcom/Default.aspx) for locations closest to the sample sites. Two averages were calculated: mean April values, and mean spawning time values. Mean temperatures and salinities for the month of April were used in order to give an estimate of how temperatures varied between sites at a fixed time-point. April was chosen as the representative month as spawning took place from February through to July. In addition, mean temperature and salinity values were obtained for a two-week period spanning the spawning date, with the sampling date taken as the midpoint and one week either side to allow for some degree of error.Mean sea temperature and mean salinity were calculated for 5–10 m of depth from Helcom data .(i) Amino acid and nucleotide diversityIn order to search for correlations between population amino acid or nucleotide diversity  Amino acid and nucleotide distanceIn order to search for correlations between genetic distance and environmental distance, Mantel’s and partial Mantel’s tests were implemented in R . GeneticIn total, 98 mtDNA genomes were fully sequenced, each comprised 16,700 base pairs, which corresponded to 3,808 amino acids when all 13 transcribed genes were concatenated. No identical sequences were encountered. Annotated genomes were submitted to GenBank (accession numbers: KC193680 - KC193777). Basic information for each coding gene is included in Additional file (i) Whole genome - The jModelTest results for all analyses can be found in Additional file (ii) Each gene individually and control region – Some Bayesian trees for individual coding genes  showed no resolution. However all other genes and the control region showed clustering that was consistent with the three main clades identified in the whole genome tree .(iii) All genes concatenated - The partitioned Bayesian nucleotide tree showed identical major clades to the whole genome Bayesian tree. The maximum likelihood tree based on amino acids for all coding genes showed no well supported clades , though the samples tended to cluster similarly as in the nucleotide-based trees. The neighbour-joining tree using between site amino acid distances showed no obvious clades, and no clear geographic structuring. However, DE-RUGEN appears to stand out at the root of the tree Figure . The mulC. harengus sequences was 2.82 Mya (95% Highest Posterior Density (HPD): 1.95 - 3.67), and between subclades 2 and 3 the mean TMRCA was 1.97 Mya (95% HPD: 1.37 – 2.64). The posterior estimate of the mean rate of evolutionary change, for the concatenated gene set over the whole tree, was 0.0047 substitutions/site/Myr (95% HPD: 0.0035 – 0.0060), with little rate variation across temporal scales within the Clupea species lineages (results not shown). Given the large uncertainty in the realized rate variation across temporal scales, which a growing body of studies suggests increases from 2 to 6 X in comparisons between within population and interspecific levels [C. harengus clade is 282 Kya, and for the two subclades 197 Kya). Analysis of the posterior distributions for the constrained nodes indicates that the TMRCA age estimates are highly influenced by the prior constraints, indicating the need for more detailed study of age estimates for these clades and the Clupeiformes as a whole before robust temporal insights can be obtained.The mean TMRCA for the  levels -59, we aST = 0.036 (P = 0.022 ± 0.004). Looking at individual coding genes and the control region, overall ΦST across populations ranged from 0 to 0.105, with ATP8 showing the lowest and COX2 the highest value , and borderline significant correlations between ATP8 amino acid distances  and ATP6 nucleotide distances  with distance by sea. No further statistically significant isolation by distance results were detected  changes in amino acid physiochemical properties in many parts of the herring mitochondrial genome Figure . The higST estimates indicated significant population structuring between many population pairs, we proceeded with correlation tests between genetic and environmental diversity.As our pairwise Φ(i) Amino acid and nucleotide diversity1,8 = 6.349, P = 0.040), ATP8 nucleotide diversity with spawning temperature  and with the interaction term spawning temperature x salinity , and COX3 amino acid diversity with spawning temperature . However, on inspection of the data, the ATP6 and COX3 amino acid diversity for the majority of the sites  is zero. No correlations were significant after the application of sequential Bonferroni correction or at a false discovery rate of 0.05.The general linear models indicated little or no correlation between genetic diversity and any of the environmental variables used  Amino acid and nucleotide distanceThe Mantel tests correlating genetic distance with environmental distance showed significant associations between the following: ATP6 nucleotide distance and spawning salinity , ATP8 nucleotide distance and spawning salinity , ATP8 amino acid distance and April salinity , COX1 amino acid distance and spawning salinity , COX1 amino acid distance and April salinity , ND3 nucleotide distance and spawning temperature  as well as latitude , and ND4L nucleotide distance and spawning temperature , and when considering amino acids, three sampling sites were divergent . Significant evidence for positive selection was found, particularly in the ND2, ND4, ND5 and ATP8 genes. Amino acid changes associated with selection were found at the base of the major clades clades, and occur more often in the three divergent sampling sites. Despite the evidence for selection, there was little evidence for correlations between genetic diversity or differentiation with environmental factors.Our results show that there are three well-supported clades of Atlantic herring in the Baltic Sea region, dating to a time long before the formation of the Baltic Sea. The three clades are all found throughout the Baltic Sea, and also at a site in the Barents Sea, with no obvious geographical clustering. Isolation by distance tests uncovered only borderline significant results for the ATP6 and ATP8 genes, before correcting for multiple testing. Despite this lack of geographical clustering, there was evidence for significant population structuring  lineages around the North American and Asian Pacific coastlines, which is of a similar order of magnitude to the dates in our study.The three main clades were well supported, and a similar pattern of clustering was found within all genes with adequate resolution. Using interspecific calibrations, the last divergence among these clades (i.e. the split between clades 2 and 3) occurred around 1.37 Mya. In order to provide a robust lower limit to this last divergence event, we also used a 10X faster rate of molecular evolution, which puts the lower limit to 137 Kya years ago. While there is a large degree of uncertainty regarding our temporal estimations of these clades, given that the Baltic Sea is a post-glacial environment that is roughly 8,000 years old , the thru et al.  estimatePungitius pungitius; [Macoma balthica) revealed three major lineages within the Baltic Sea and North Sea, with one clade occurring only within the Baltic Sea and Alaska, a second clade occurring predominantly outside the Baltic Sea, and a third clade found in both the Baltic and North Seas; these lineages also diverged long before the formation of the Baltic Sea, at around 9.8-39 Mya [Multiple genetic lineages seem to be common in the Baltic region in a number of species. For example, the ninespine stickleback  apparen-39 Mya . It is cGadus morhua) found multiple clades but with little clear geographical patterning, despite their four sampling locations spanning the North Atlantic [C. pallasii) did manage to detect population structure around the North American and Asian Pacific coastlines [Similarly to our study, a whole mitochondrial genome study of the cod , largerCorrelations between genetic diversity, differentiation and environmental factors were also of interest due to the strong environmental gradients in the Baltic Sea, as well as the potential adaptive value of mitochondrial variability. There was no evidence that genetic diversity (either nucleotide or amino acid) was correlated with distance from the entrance to the Baltic Sea, latitude, salinity, or temperature. However, there was some evidence that genetic distances at the COX1 gene are associated with salinity, and genetic distances at the ND3 and ND4L genes (both are members of the NADH Complex 1) are associated with temperature at spawning time, after correcting for the effects of geographical distance. ND3 genetic distance was also associated with latitude, which is likely to be due to the variation in temperature at different latitudes rather than a spatial effect, as there was no indication of isolation by distance in this gene. None of these correlations remained significant after the application of corrections for multiple testing, however, these corrections are very conservative particularly as the α values become very low given the number of tests that we have performed, and there is much debate regarding the application of such tests . The tiAnacystis nidulans[Synechococcus 6311 cells grown in a saline environment show a tenfold increase in cytochrome oxidase activity [There is very little published material on the adaptive roles of individual mitochondrial genes. However, there are indications that cytochrome oxidase enzyme activity increases in high salinity environments in the cyanobacterium  nidulans, and in ctivity . In addictivity . AlthougFrom the perspective of fisheries management, this work shows that although there are distinct mitochondrial lineages of herring in the Baltic Sea, the Atlantic herring management units in the Baltic region cannot be defined on the basis of these lineages due to a lack of clear geographical structuring. However, our data does indicate higher levels of population differentiation than expected at random, with some populations appearing particularly divergent when looking at amino acid data. This divergence aligns with the within-Baltic genetic structure described using transcriptome-derived microsatellite markers .There is also some indication that positive selection has occurred on the amino acids in the divergent populations. Furthermore, transcriptome-derived microsatellites showed evidence for local adaptation to temperature and selection , and a rThe development of molecular markers located in selected regions of genomes has gained attention in the conservation genomics literature, the argument being that such markers may be able to play a large role in management by helping to identify management or conservation units . The mitAll authors declare that they have no competing interest.AGFT carried out the molecular genetics lab work, performed all analyses except for clade dating, and wrote the paper. CWW performed the dating analysis. All authors contributed to the conception and design of the study, and contributed to drafting the final manuscript. All authors read and approved the final manuscript.Information on samples. SD= fisheries subdivision, n = number of individuals. The latitude and longitude for each site are given to the accuracy provided by the collectors.Click here for filePrimer details. Primer details including name, sequence, annealing temperature (Ta), GC content, and the length of the amplified product (in base pairs).Click here for fileBasic information and jModelTest results for whole genome and each gene. This table shows the complex, length (in base pairs), number of segregating sites (S), model chosen by the Bayesian Information Criterion (BIC), nucleotide frequencies, substitution frequencies (R), proportion of invariable sites (p-inv), gamma shape, and transition/transversion ratio (ti/tv) for the whole genome and each gene individually.Click here for fileAMOVA results for whole genome, individual coding genes, and the control region (CR). Presented are the ΦST , the probability that the observed data is within the range of the random data (p(rand=obs)), the probability of randomly getting an FST that is higher than the observed data (P(rand>obs)), and the overall probability (P). On application of sequential Bonferroni correction, or a false discovery rate of 0.05, only the AMOVA for COX2 remains significant.Click here for filePopulation-specific ΦST indices from the AMOVA analyses. Population-specific ΦST indices from the AMOVA analyses, representing the degree of evolution of a particular population from a common ancestral population. These indicate whether certain populations contribute differently to others to the average ΦST.Click here for filePairwise ΦST for (a) the whole genome and (b) the COX2 gene. Pairwise ΦST for (a) the whole genome and (b) the COX2 gene. The COX2 gene showed significant population structure in the AMOVA analysis. The numbers below the diagonals are ΦST and the numbers above the diagonals are the probability values. The probability values shown in bold are <0.05.Click here for fileIsolation by distance results. The Pearson product–moment correlation coefficient statistic (r) and the probability values (P) are shown for whole genome (Genome), all genes concatenated (Genes), each individual coding gene, and the control region (CR). Separate results are shown for nucleotide and amino acid data. There is no amino acid data shown for the whole genome as large parts of the genome are non-coding, nor for the control region. In addition, amino acid results are not shown for the ND1 gene as there was no variation. Significant results are shown in bold (p<0.05). All tests were non-significant after the application of sequential Bonferroni correction or at a false discovery rate of 0.05; the corrections were applied separately for nucleotides and amino acids.Click here for fileTreeSAAP results summary. Showing the total number of codons for each coding gene and complex, the number of these codons that were detected as being under significant positive disruptive selection (P ≤ 0.001), and the proportion of codons under selection. For each sampling site we show the number of individuals sampled, the number of codons under significant positive disruptive selection , and the number of codons corrected for number of individuals.Click here for fileResults of general linear models. The response variables were the genetic diversity measures, either nucleotide diversity (nuc) or amino acid diversity (aa), while the explanatory variables were distance from the entrance to the Baltic (Distance), latitude, temperature and salinity at spawning time, temperature and salinity for April, and the interactions between temperature and salinity at spawning time and for April. F statistics ) and probability values (P) are given. P values <0.05 are shown in bold. No correlations were significant after the application of sequential Bonferroni correction or at a False Discovery Rate of 0.05; the corrections were applied within each environmental variable, and separately for nucleotides and amino acids.Click here for fileResults of Mantel and partial Mantel tests correlating genetic distance with environmental distance. The Pearson product–moment correlation coefficient statistic (r) and the probability values (P) are shown for whole genome (Genome), all genes concatenated (Genes), each individual coding gene, and the control region (CR). Separate results are shown for nucleotide and amino acid data. There is no amino acid data shown for the whole genome as large parts of the genome are non-coding, nor for the control region. In addition, amino acid results are not shown for the ND1 gene as there was no variation. Partial Mantel tests were all performed using shortest geographical distance by sea as the conditioning variable. Significant (p<0.05) results are shown in bold. No correlations were significant after the application of sequential Bonferroni correction or at a false discovery rate of 0.05; the corrections were applied within each environmental variable, separately for nucleotides and amino acids, and separately for Mantel and partial Mantel tests.Click here for file


This study aims to describe the trends in and determinants of six month mortality and loss to follow up (LTFU) during 2005–2009 in 13 outpatient clinics in Vietnam.Data were obtained from clinical records of 3,449 Vietnamese HIV/AIDS patients aged 18 years or older who initiated ART between 1 January 2005 and 31 December 2009. Mantel-Haenszel chi-square test, log rank test were conducted to examine the trends of baseline characteristics, six month mortality and LTFU. Cox proportional hazards regression models were performed to compute hazard ratio (HR) and 95% Confidence Interval (CI).3 versus <50 cells/mm3 0.26, 95%CI: 0.18–0.52), low baseline BMI , co-infection with TB , history of injecting drugs . Characteristics associated with LTFU were younger age , males , and poor adherence .Though there was a declining trend, the incidence of six month mortality and LTFU remained as high as 6% and 15%, respectively. Characteristics associated with six month mortality were gender , years of initiation , low baseline CD4 (HR 350–500 cells/mmTo reduce early mortality, special attention is required to ensure timely access to ART services, particularly for patients at higher risk. Patients at risk for LTFU after ART initiation should be targeted through enhancing treatment counselling and improving patient tracing system at ART clinics. Loss to follow up (LTFU) and mortality after initiation of antiretroviral therapy (ART) are commonly used outcomes to evaluate the effectiveness of ART and to identify opportunities for program improvement Despite significant expansion and increased coverage of HIV care and treatment in Vietnam, high rates of LTFU  have been reported LTFU and early mortality present key challenges in the implementation of ART programmes. High rates of early mortality, in particular, can reflect a high level of treatment failure, drug resistance, or poor adherence to treatment regimens A prospective study design was used. The complete description of cohort participants and the selection of clinics has been previously published In the present analysis, outcome measures were deaths occurring in the first six months of ART and patients who were identified as LTFU during the entire follow up period. Patients were defined as LTFU based on the patient records: if they had started ART and were absent from the clinic for more than three months and did not return to the clinics by the end of the study period (30 November 2010).Independent variables included: baseline age (expressed in years) and gender; transmission routes ; baseline clinical characteristics ; adherence to treatment every six months following ART initiation; and the  year of ART initiation. Adherence was designated as “Good” if patient had >95% self-reported adherence rate in the previous month, and “Poor” if the patient had <95%.Descriptive statistics were performed to derive means and standard deviations for continuous variables  and to provide frequency distributions for categorical variables . Difference in trend of proportion of baseline WHO stage, baseline OI, baseline CTX, baseline TB by initiation years were assessed by Mantel-Haenszel chi-square test. Differences in trend of six month mortality and LTFU by initiation years were assessed using log rank test. The relationships between baseline characteristics and outcomes were tested using proportional hazards regression models. Crude and adjusted hazard ratios (HR) and 95% Confidence Intervals (CI) were reported. All analyses were performed using SAS (version 9.2).p<0.25), and these variables were subsequently fitted into the initial multivariate Cox regression models. Among variables that were not significantly associated with the outcome (p>0.05), the one that was least significant  was removed and the model refitted with the remaining variables. This procedure was repeated in a backward stepwise manner. In the final model, only variables that were significantly associated with the outcome (p≤0.05) were retained.The analysis began with univariate analysis to identify characteristics that were likely to be associated with the outcome . The mean age at baseline was 30 years old, 65% were infected through injecting drugs. After five years of follow up, 198 (5.7%) patients have died within first six months on ART, 530 (15.4%) patients were lost to follow up. The descriptive information of study participants in the cohort, the patients died in the first six months of ART, and the LTFU are presented in The current analysis involved 3,449 patients, 75% being males, who initiated ART between 13 (SD: 45) in 2005 to 101 (SD: 86) cells/mm3 in 2009 (simple linear regression p = 0.001). The proportion of patients at Stage IV decreased from 61% in 2005 to 39% in 2009 (Mantel-Haenszel chi-square test p = 0.01). However, there was no significant trend in the proportion of patients with baseline OI (Mantel-Haenszel p = 0.43), TB co-infection (Mantel-Haenszel p = 0.51), and baseline CTX use (Mantel-Haenszel p = 0.73). As presented in As summarised in 3 was significantly associated with a 67% lower risk of death in the first 6 months after ART initiation, compared with having a baseline CD4 count below 50 cells/mm3 .Table 3 shows that there is a strong association between year of initiation and the risk of six month mortality. After adjustment for other covariates, being enrolled in 2009 was significantly associated with a 46% lower risk of death within the first 6 months, compared with being initiated in 2005 . Also, having a baseline CD4 cell count of 200–350 cells/mmThe risk of six month mortality was significantly lower for females , but higher for being infected via injecting drugs , having TB at initiation , or being at a more advanced WHO clinical stage . Furthermore, for every unit increase in BMI, the risk of early mortality reduced by 4% .Baseline age , opportunistic infection , baseline Cotrimoxazol use , and adherence in the first six months  were not significantly associated with mortality in the first six months after ART initiation.In both univariate and multivariate models, younger patients were more likely to be LTFU than older patients . The risk of LTFU was significantly lower for females than for males , but was higher for those infected via IDU  and those who had TB at ART initiation . Being enrolled in 2009 was significantly associated with a lower risk of LTFU, compared with being enrolled in 2005 . Poor adherence was also significantly associated with LTFU in the same direction .3, 0.56, 95%CI 0.28–1.12), and baseline BMI  were not significantly associated with LTFU over the entire study period.Baseline Cotrimoxazol use , baseline WHO clinical stage , baseline CD4 . More importantly, earlier studies in Vietnam and elsewhere p<0.001) (data not shown).Younger age was the only demographic characteristic that predicted LTFU. This finding is consistent with a number of studies in Eastern, Southern, and Western Africa Our study has several limitations. First, LTFU patients may have been identified simply on the basis of persistent absence from clinic appointments without tracing patients who have died without notification to the clinic. A recent study in South Africa, for example, found that 20–60% of patients LTFU have died during the follow up Second, data available for the present analysis were based on clinical records that were entirely dependent on the discretion of the responsible clinic staff leading to missing values in a number of variables. However, the missing values are likely to have minor impacts on the findings as they accounted for less than 5%. Further, as human resource shortages were reported in many clinics during the study period Third, the definition of LTFU (no attendance within three months of the last scheduled visit) may have resulted in misclassification between treatment interruption and LTFU given some patients may have returned to a clinic after three months. Further analysis, however, showed that only 19 (0.6%) patients returned to a clinic more than three months after their last scheduled visit which clearly indicates that the impact of any such misclassification on the overall findings would be minimal. Similarly, the transfer of patients between ART clinics is unlikely to have resulted in them being misclassified as LTFU because all transfers require a referral letter from the old to the new clinic and details of the transfer must be recorded in patient records.Fourth, the quality of the services provided by outpatient clinics, which may have a significant impact on LTFU and mortality, were not investigated in our study. Finally, the extent to which the findings are generalizable to all of Vietnam is unclear, given this study comprised 13 clinics from five provinces. Despite these shortcomings, a robust and consistent data protocol was adhered to which would minimize coding issues.Findings from this study suggest that the rapid expansion of the national ART programme in Vietnam has produced positive impacts. This was reflected in more patients being enrolled at earlier clinical stages with higher baseline CD4 counts, and decreased mortality and LTFU. However, the incidence of early mortality and LTFU remained significant and could benefit from further system and programmatic improvement. To reduce early mortality, special attention is required to ensure timely access to ART services. Loss to follow up also requires improvements after ART initiation. People who start ART with lower adherence, young people, and injecting drug users should be targeted through improving treatment counselling and the patient tracing system at ART clinics. Given that the number of HIV patients receiving ART in Vietnam will continue to increase resulting from the less stringent initiation criteria, additional studies are also needed to identify effective public health and medical interventions to reduce mortality and LTFU as well as to improve treatment outcomes, especially for the high-risk patient groups.