Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp. and Temora sp. from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.     

The effect of induced heat waves on Pinus taeda and Quercus rubra seedlings in ambient and elevated CO(2) atmospheres

Here, we investigated the effect of different heat-wave intensities applied at two atmospheric CO(2) concentrations ([CO(2) ]) on seedlings of two tree species, loblolly pine (Pinus taeda) and northern red oak (Quercus rubra). Seedlings were assigned to treatment combinations of two levels of [CO(2) ] (380 or 700?μmol?mol(-1) ) and four levels of air temperature (ambient, ambient +3°C, or 7-d heat waves consisting of a biweekly +6°C heat wave, or a monthly +12°C heat wave). Treatments were maintained throughout the growing season, thus receiving equal heat sums. We measured gas exchange and fluorescence parameters before, during and after a mid-summer heat wave. The +12°C heat wave, significantly reduced net photosynthesis (A(net) ) in both species and [CO(2) ] treatments but this effect was diminished in elevated [CO(2) ]. The decrease in A(net) was accompanied by a decrease in F(v) '/F(m) ' in P.?taeda and Φ(PSII) in Q.?rubra. Our findings suggest that, if soil moisture is adequate, trees will experience negative effects in photosynthetic performance only with the occurrence of extreme heat waves. As elevated [CO(2) ] diminished these negative effects, the future climate may not be as detrimental to plant communities as previously assumed.     

Is Rumination after Bereavement Linked with Loss Avoidance? Evidence from Eye-Tracking

Rumination is a risk factor in adjustment to bereavement. It is associated with and predicts psychopathology after loss. Yet, the function of rumination in bereavement remains unclear. In the past, researchers often assumed rumination to be a maladaptive confrontation process. However, based on cognitive avoidance theories of worry in generalised anxiety disorder (GAD) and rumination after post-traumatic stress disorder (PTSD), others have suggested that rumination may serve to avoid painful aspects of the loss, thereby contributing to complicated grief. To examine if rumination is linked with loss avoidance, an eye-tracking study was conducted with 54 bereaved individuals (27 high and 27 low ruminators). On 24 trials, participants looked for 10 seconds at a picture of the deceased and a picture of a stranger, randomly combined with negative, neutral or loss-related words. High ruminators were expected to show initial vigilance followed by subsequent disengagement for loss stimuli (i.e., picture deceased with a loss word) in the first 1500 ms. Additionally, we expected high ruminators to avoid these loss stimuli and to show attentional preference for non-loss-related negative stimuli (i.e., picture stranger with a negative word) on longer exposure durations (1500–10000 ms). Contrary to expectations, we found no evidence for an effect of rumination on vigilance and disengagement of loss stimuli in the first 1500 ms. However, in the 1500–10000 ms interval, high ruminators showed shorter gaze times for loss stimuli and longer gaze times for negative (and neutral) non-loss-related stimuli, even when controlling for depression and complicated grief symptom levels. Effects of rumination on average fixation times mirrored these findings. This suggests that rumination and loss avoidance are closely associated. A potential clinical implication is that rumination and grief complications after bereavement may be reduced through the use of exposure and acceptance-based therapeutic techniques.     

The role of N-glycosylation in function and surface trafficking of the human dopamine transporter

The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.     

Mortality among Human Immunodeficiency Virus Type 2-Positive Villagers in Rural Guinea-Bissau Is Correlated with Viral Genotype

We present the results of a 6-year study of 131 human immunodeficiency virus (HIV) type 2 (HIV-2)-infected individuals from a rural population in Guinea-Bissau. Proviral DNA sequences 1.3 kb in length were obtained from each individual and, together with clinical data, including proviral load and CD4 and CD8 levels, were used to assess whether viral genotype influences clinical outcome. With a phylogenetic model, a correlation was found between viral genotype and mortality; this correlation was not due to confounding factors, such as age-specific viral strains or cohabitation of patients. The data provide strong evidence for the involvement of viral genetic factors in determining HIV disease progression in vivo. The pattern of association found suggests that virulence factors are multiple and scattered throughout the HIV-2 genome and can be rapidly gained or lost by the virus through a combination of mutation and recombination. These findings may lead to the identification of viral determinants of HIV disease progression.     

Differential responses of cellular immunity in patients undergoing neoadjuvant therapy followed by surgery for carcinoma of the oesophagus

Background  To compare immune responses following neoadjuvant chemoradiation therapy in combination with hyperthermia plus surgery to those induced by surgery alone in patients with oesophageal cancer. Methods  Thirty-two patients with histopathologically proven oesophageal cancer, scheduled for potentially curative transhiatal or transthoracic oesophagectomy with (neo, n = 20) or without (control, n = 12) neoadjuvant thermochemoradiation therapy (ThCR) were included. Peripheral blood samples were obtained before ThCR, after 2 weeks of ThCR, 1 day before surgery, on postoperative days 1, 3, 7, and 6 weeks after surgery, for white blood cell counts, lymphocyte subsets and T helper type 1 (Th1) and type 2 (Th2) lymphocyte responses. Results  Neo patients showed a significant decrease in granulocytes and lymphocyte subsets, and T cell cytokines after 2 weeks of ThCR. Only CD8+ (cytotoxic) T cells recovered after ThCR to reach normal levels prior to surgery. In contrast, CD4+ T (helper) cells, and NK- and B cells in neo patients did not recover prior to surgery (all P < 0.05). Oesophagectomy induced a significant increase in granulocytes and a decrease in lymphocytes (and subsets). Only those subsets that had not recovered after ThCR (CD4+ T cells, NK and B cells but not CD8+ T cells), were significantly lower (all P < 0.05) during the entire postoperative study period. Postoperatively, the stimulated cytokine production capacity of Th1 and Th2 cells, corrected for number of T cells, was not significantly different between the groups. Conclusion  Neoadjuvant thermochemoradiation for oesophageal cancer caused significant disturbances of host cellular immunity with reduced T, NK and B cell counts, and differential recovery of cytotoxic and helper T cells leading to prolonged T cell imbalance that extends beyond the time of surgery. The functional and anti-tumour consequences of this immunodisturbance need further investigation, as recovery of T helper cytokine production towards surgery was less impaired than T helper cell counts.     

MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides

MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.     

Successful 96- and 144-hour experimental kidney preservation: A combination of standard machine preservation and newly developed normothermic ex vivo perfusion

In order to enable time-consuming matching and selection procedures (PLT, MLC) in kidney transplantation, a new approach to prolonged kidney preservation was studied. In a canine autotransplantation and contralateral nephrectomy model hypothermic machine preservation was interrupted by a short period of ex vivo perfusion on the donor animal. In a 96-hr experiment post-transplantation serum creatinines were significantly lower in the ex vivo perfused group versus the control group. All animals survived. In a 144-hr study five out of six survived in the ex vivo perfused group as opposed to one out of six in the control group. The beneficial effect of this ex vivo perfusion in the course of hypothermic machine preservation may be due either to a washout of metabolic waste products or to a restoration of exhausted enzyme systems.