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TIM23-mediated insertion of transmembrane alpha-helices into the mitochondrial inner membrane
(2011)
Botelho, Salomé Calado ; Österberg, Marie ; Reichert, Andreas ; Yamano, Koji ; Björkholm, Patrik ; Endo, Toshiya ; Heijne, Gunnar von ; Kim, Hyun
While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) alpha-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems. Keywords: CoxVa , membrane protein , Mgm1p , mitochondria , TIM23
Pfeilschifter, Waltraud ; Czech-Zechmeister, Bozena ; Sujak, Marian ; Förch, Christian ; Wichelhaus, Thomas Alexander ; Pfeilschifter, Josef
Background: FTY720, an immunomodulator derived from a fungal metabolite which reduces circulating lymphocyte counts by increasing the homing of lymphocytes to the lymph nodes has recently gained interest in stroke research. The aim of this study was to evaluate the protective efficacy of FTY720 in cerebral ischemia in two different application paradigms and to gather first data on the effect of FTY720 on the rate of spontaneous bacterial infections in experimental stroke. Methods: Middle cerebral artery occlusion (MCAO) in C57BL/6 mice (strain J, groups of 10 animals) was performed with two different durations of ischemia (90 min and 3 h) and FTY720 was applied 2 h after vessel occlusion to study the impact of reperfusion on the protective potency of FTY720. Lesion size was determined by TTC staining. Mice treated with FTY720 or vehicle were sacrificed 48 h after 90 min MCAO to determine the bacterial burden in lung and blood. Results: FTY720 1 mg/kg significantly reduced ischemic lesion size when administered 2 h after the onset of MCAO for 3 h (45.4 +/- 22.7 mm3 vs. 84.7 +/- 23.6 mm3 in control mice, p = 0.001) and also when administered after reperfusion, 2 h after the onset of MCAO for 90 min (31.1 +/- 28.49 mm3 vs. 69.6 +/- 27.2 mm3 in control mice, p = 0.013). Bacterial burden of lung homogenates 48 h after stroke did not increase in the group treated with the immunomodulator FTY720 while there was no spontaneous bacteremia 48 h after MCAO in treated and untreated animals. Conclusions: Our results corroborate the experimental evidence of the protective effect of FTY720 seen in different rodent stroke models. Interestingly, we found no increase in bacterial lung infections even though FTY720 strongly reduces the number of circulating leukocytes.
NK cells are part of the innate immune system, and are important players in the body’s first defence line against virus-infected and malignantly transformed cells. While T cells recognize neoplastic cells in an MHC-restricted fashion, NK cells do not require prior sensitization and education about the target. In leukemia and lymphoma patients undergoing allogeneic hematopoietic stem cell transplantation not only T cells but also NK cells have been found to mediate potent graft-versus-tumor effects. Hence, autologous or donor-derived NK cells hold great promise for cancer immunotherapy. Since the generation of highly purified NK cell products for clinical applications is labor-intensive and time consuming, established human NK cell lines such as NK-92 are also being considered for clinical protocols. NK-92 cells display phenotypic and functional characteristics similar to activated primary NK cells. While NK-92 cells are highly cytotoxic towards malignant cells of hematologic origin, they do not affect healthy human tissues. NK-92 cells can be expanded under GMP-compliant conditions, and can therefore be provided in sufficient numbers with defined phenotypic characteristics for clinical applications. Safety of NK-92 cells for adoptive immunotherapy was already shown in two phase I/II clinical trials...
Melfsen, Siebke ; Kühnemund, Martina ; Schwieger, Judith ; Warnke, Andreas ; Stadler, Christina ; Poustka, Fritz ; Stangier, Ulrich
Background: Although literature provides support for cognitive behavioral therapy (CBT) as an efficacious intervention for social phobia, more research is needed to improve treatments for children. Methods: Forty four Caucasian children (ages 8-14) meeting diagnostic criteria of social phobia according to the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; APA, 1994) were randomly allocated to either a newly developed CBT program focusing on cognition according to the model of Clark and Wells (n = 21) or a wait-list control group (n = 23). The primary outcome measure was clinical improvement. Secondary outcomes included improvements in anxiety coping, dysfunctional cognitions, interaction frequency and comorbid symptoms. Outcome measures included child report and clinican completed measures as well as a diagnostic interview. Results: Significant differences between treatment participants (4 dropouts) and controls (2 dropouts) were observed at post test on the German version of the Social Phobia and Anxiety Inventory for Children. Furthermore, in the treatment group, significantly more children were free of diagnosis than in wait-list group at post-test. Additional child completed and clinician completed measures support the results. Discussion: The study is a first step towards investigating whether CBT focusing on cognition is efficacious in treating children with social phobia. Future research will need to compare this treatment to an active treatment group. There remain the questions of whether the effect of the treatment is specific to the disorder and whether the underlying theoretical model is adequate. Conclusion: Preliminary support is provided for the efficacy of the cognitive behavioral treatment focusing on cognition in socially phobic children. Active comparators should be established with other evidence-based CBT programs for anxiety disorders, which differ significantly in their dosage and type of cognitive interventions from those of the manual under evaluation (e.g. Coping Cat).
Purpose of the Study: The purpose of the current study was to evaluate the role of radiofrequency (RF) and microwave (MW) ablation in the treatment of pulmonary neoplasms. Materials and Methods: From March 2004 to January 2009, 164 patients (92 males, 72 females; mean age 59.7 years, SD: 10.2) underwent computed tomography (CT)-guided percutaneous RFA of pulmonary malignancies. RFA was performed on 248 lung lesions (20 primary lesions and 228 metastatic lesions) in 248 sessions (one lesion per session). Tumors were pathologically proven and were classified as primary lung neoplasms in 20 patients (non-small cell lung cancer) and as metastatic lung neoplasms in 144 patients. RFA was performed using: a) CelonProSurge bipolar internally cooled applicator b) RITA®StarburstTMXL. From December 2007 to October 2009, 80 patients (30 males, 50 females; mean age 59.7 years, range: 48-68, SD: 6.4) underwent computed tomography (CT) guided percutaneous MW ablation of pulmonary metastases from variable histopathological primaries. MW was performed on 130 lung lesions in 130 sessions (one lesion per session) using Valleylab TM system. Results: The overall success rate of RFA was 67.7% (168/248 lesions), with overall failure rate either due to tumor residue or recurrence on follow up in 32.3% (80/248) with mean time to tumor progress was 5.6 months SD: 2.99 (Range:1-18 months). Complete successful ablation was achieved in patients treated by MWA in 73.1% (95/130 lesions), with failure rate either due to tumor residue or recurrence on follow up in 26.9% (35/130) with mean time to tumor progress 6 months SD: 2.83 (Range:1-12months). Correlation of the histopathological type of the lesion and the end result of ablation therapy revealed insignificant correlation in both RFA and MWA (p > 0.1). The preablation tumor size was one of the most significant factors that determined the end result of ablation. In RFA successful tumor ablation was significant statistically for lesions with maximal axial diameter up to 2.5 cm (110/140) in comparison to lesions of more than 2.5 cm in maximal axial diameter (58/108) (Fisher’s exact test: p < 0.0001). While in MW ablated lesions successful tumor ablation was significant statistically for lesions with maximal axial diameter up to 3 cm (90/110) in comparison to lesions of more than 3 cm in maximal axial diameter (5/20) (Fisher’s exact test: p < 0.001). The location of the lesion was another important factor that determined the end result of ablation. In both RFA and MWA successful ablation was significantly more correlated to peripheral lesions (RFA: 120/160, 80% / MWA: 80/100, 80%) than centrally located lesions (RFA: 48/88, 50%; MWA: 15/30, 50%) (Fisher’s Exact Test: p > 0.001). For successfully RFA ablated cases mean preablation tumor volumes 1.9 cc SD: 0.9 (range: 0.3 - 4.25 cc) while for failed cases the mean tumor volume was 3.7 SD: 2.4 (range: 0.8 – 6.8cc). For successfully MW ablated cases the mean preablation tumor volume: 2.4 cc SD: 2.2 (range: 0.25-8.2 cc) while for failed cases the mean tumor volume was 3.5 SD: 2.6 (range: 0.3 – 7.1 cc). In RFA the survival rates at 12, 24 and 36 months were 90%, 78% and 68% respectively while in MWA treated patients the survival rate within 12 months follow up period was 96% while at 20 month the survival rate was 77%. Complications associated with the ablation therapy were: a) procedure related mortality: 0.4% (1/248) in RFA due to massive pulmonary hemorrhage versus 0% (0/130) in MWA, b) pneumothorax: 11.3% (28/240) in RFA versus 8.5% (11/130) in MWA, c) pulmonary Hemorrhage: 17.7% (44 of 248 sessions) of which one patient had massive uncontrolled bleeding and immediate death versus 6.2% (8/130) in MWA, d) pleural effusion: 3.2 % (8 of 248 sessions) in RFA versus 3.8 % (6/130) in MWA, e) hemoptysis: 4% (10/248) in RFA versus 4.6% (6/130) in MWA ranging from mild tinged sputum to frank bleeding, f) infection: 0.4% (1/248) in RFA, versus 0% in MWA, and g) post ablation pain: 10% (25/248) in RFA versus 9.2% (12/130) in MWA. Pain was generally adequately controlled by analgesics. Conclusion: Radiofrequency and microwave ablation are effective minimally invasive tools and may be safely applied for management of lung malignancy. The success of ablation therapy is significantly correlated to the preablation tumor size, volume and tumor location.
Wiesmann, Frank ; Vachta, Jan ; Ehret, Robert ; Walter, Hauke ; Kaiser, Rolf ; Stürmer, Martin ; Tappe, André ; Däumer, Martin ; Berg, Thomas ; Naeth, Gudrun ; Braun, Patrick ; Knechten, Heribert
Background: Although being considered as a rarely observed HIV-1 protease mutation in clinical isolates, the L76V-prevalence increased 1998-2008 in some European countries most likely due to the approval of Lopinavir, Amprenavir and Darunavir which can select L76V. Beside an enhancement of resistance, L76V is also discussed to confer hypersusceptibility to the drugs Atazanavir and Saquinavir which might enable new treatment strategies by trying to take advantage of particular mutations. Results: Based on a cohort of 47 L76V-positive patients, we examined if there might exist a clinical advantage for L76V-positive patients concerning long-term success of PI-containing regimens in patients with limited therapy options. Genotypic- and phenotypic HIV-resistance tests from 47 mostly multi-resistant, L76V-positive patients throughout Germany were accomplished retrospectively 1999-2009. Five genotype-based drug-susceptibility predictions received from online interpretation-tools for Atazanavir, Saquinavir, Amprenavir and Lopinavir, were compared to phenotype-based predictions that were determined by using a recombinant virus assay along with a Virtual Phenotype™(Virco). The clinical outcome of the L76V-adapted follow-up therapy was determined by monitoring viral load for 96 weeks. Conclusions: In this analysis, the mostly used interpretation systems overestimated the L76V-mutation concerning Atazanavir- and SQV resistance. In fact, a clear benefit in drug susceptibility for these drugs was observed in phenotype analysis after establishment of L76V. More importantly, long-term therapy success was significantly higher in patients receiving Atazanavir and/or Saquinavir plus one L76V-selecting drug compared to patients without L76V-selecting agents (p = 0.002). In case of L76V-occurrence ATV and/or SQV may represent encouraging options for patients in deep salvage situations.
Vacuum-assisted closure (VAC) of complex infected wounds has recently gained popularity among various surgical specialties. The system is based on the application of negative pressure by controlled suction to the wound surface. The effectiveness of the VAC System on microcirculation and the promotion of granulation tissue proliferation are proved. In our case report we illustrate a scenario were a patient developed severe bleeding from the ascending aorta by penetration of wire fragments in the vessel. We conclude that all free particles in the sternum have to be removed completely before negative pressure is used.
Summary: Information and communication is critical to the successful management of infectious diseases because an effective communication strategy prevents the surge of anxious patients who have not been genuinely exposed to the pathogen ('low risk patients') affecting medical infrastructures (1) and the future transmission of the infectious agent (2). Surge of low risk patients: The arrival of large numbers of low risk patients at hospitals following an infectious diseases emergency would be problematic for three main reasons. First, it would complicate the situation at hospitals receiving exposed patients, delaying the treatment of the acutely ill, creating difficulties of crowd control and tying up medical resources. Second, for the low risk patients themselves, attending hospital following an infectious disease emergency might increase their risk of exposure to the agent in question. Third, the needs of low risk patients may be poorly attended to at hospitals which are already overstretched dealing with medical casualties. Future transmission: Obtaining early information about symptoms and isolating infected patients is the most effective strategy to interrupt the chain of infection in the public in the absence of specific prophylaxis or treatment. Particularly at the beginning of an outbreak, these nonpharmaceutical interventions play an important role in enabling the early detection of signs or symptoms and in encouraging passengers to adopt appropriate preventive behaviour in order to limit the spread of the disease. This thesis includes two papers dealing with this problem: The first part is a systemic literature review of information needs following an infectious disease emergency (Anthrax, SARS, Pneumonic Plague). The key question was: what are the information needs of the public during an infectious disease emergency? The second part is an empirical investigation of information needs and communication strategies at the airport during the early stage of the Influenza Pandemic. The key question here was: what communication strategies help to meet the information needs and to enable the public to behave appropriately and responsibly? Conclusions: Evidence from the anthrax attacks in the United States suggested that a surge of low risk patients is by no means inevitable. Data from the SARS outbreak illustrated that if hospitals are seen as sources of contagion, many patients with non-bioterrorism related health care needs may delay seeking help. Finally, the events surrounding the Pneumonic Plague outbreak of 1994 in Surat, India, highlighted the need for the public to be kept adequately informed about an incident to avoid creating rumours. Clear, consistent and credible information is key to the successful management of infectious disease outbreaks. The results of the empirical investigation suggested that the desire for information is a reflection of current anxiety and does not mirror the objective scientific assessment of exposure. The airport study showed that perceived information needs were directly related to anxiety – the least anxious did not require any further information, the most anxious reported significant information needs concerning medical treatment, public health management and the assessment of the ongoing situation – irrespective of their actual exposure. A communication strategy only focussing on the 'real' exposed individuals neglects the information needs of those worrying about having contracted the virus and seeking medical attendance. Effective communication strategies should enable the general public to detect early signs or symptoms and provide them with behaviour advice to prevent the further transmission of the infectious agent. These include the provision of clear information about the incident, the symptoms and what to do to prevent the further transmission, detailed and regularly updated information in various media formats (telephone, internet, etc.) and rapid triage at hospital entrances to guide patients to the appropriate medical infrastructures. Relevance: These research findings could contribute to a shift in the organisational and communicative approach responding to infectious diseases outbreaks and could be considered relevant for future risk communication and policy decision making.
The pathophysiology of schizophrenia is still poorly understood. Investigating the neurophysiological correlates of cognitive dysfunction with functional neuroimaging techniques such as electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) is widely considered to be a possible solution for this problem. Working memory impairment is one of the most prominent cognitive impairments found in schizophrenia. Working memory can be divided into a number of component processes, encoding, maintenance and retrieval. They appear to be differentially affected in schizophrenia, but little is known about the neurophysiological disturbances which contribute to deficits in these component processes. The aim of this dissertation was to elucidate the neurophysiological underpinnings of the component processes of working memory and their disturbance in schizophrenia. In the first study the the neurophysiological substrates of visual working memory capacity limitations were investigated during encoding, maintenance and retrieval in 12 healthy subjects using event-related fMRI. Subjects had to encode up to four abstract visual shapes and maintain them in working memory for 12 seconds. Afterwards a test stimulus was presented, which matched one of the previously shown shapes in fifty percent of the trials. A bilateral inverted U-shape pattern of BOLD activity with increasing memory load in areas closely linked with selective attention, i.e. the frontal eye fields and areas around the intraparietal sulcus, was observed already during encoding. The increase of the number of stored items from memory load three to memory load four in these regions was negatively correlated with the increase of BOLD activity from memory load three to memory load four. These results point to a crucial role of attentional processes for the limited capacity of working memory. In the second study, the contribution of early perceptual processing deficits during encoding and retrieval to working memory dysfunction was investigated in 17 patients with schizophrenia and 17 healthy control subjects using EEG and event-related fMRI. A slightly modified version of the working memory task used in the fist study was employed. Participants only had to encode and maintain up to three items. In patients the amplitude of the P1 event-related potential was significantly reduced already during encoding in all memory load conditions. Similarly, BOLD activity in early visual areas known to generate the P1 was significantly reduced in patients. In controls, a stronger P1 amplitude increase with increasing memory load predicted better performance. These findings indicate that in addition to later memory related processing stages early visual processing is disturbed in schizophrenia and contributes to working memory dysfunction by impairing the encoding of information. In the third study, which was based on the same data set as the second study, cortical activity and functional connectivity in 17 patients with schizophrenia and 17 to healthy control subjects during the working memory encoding, maintenance and retrieval was investigated using event-related fMRI. Patients had reduced working memory capacity. During encoding activation in the left ventrolateral prefrontal cortex and extrastriate visual cortex was reduced in patients but positively correlated with working memory capacity in controls. During early maintenance patients switched from hyper- to hypoactivation with increasing memory load in a fronto-parietal network which included left dorsolateral prefrontal cortex. During retrieval right ventrolateral prefrontal hyperactivation was correlated with encoding-related hypoactivation of left ventrolateral prefrontal cortex in patients. Cortical dysfunction in patients during encoding and retrieval was accompanied by abnormal functional connectivity between fronto-parietal and visual areas. These findings indicate a primary encoding deficit in patients caused by a dysfunction of prefrontal and visual areas. The findings of these studies suggest that isolating the component processes of working memory leads to more specific markers of cortical dysfunction in schizophrenia, which had been obscured in previous studies. This approach may help to identify more reliable biomarkers and endophenotypes of schizophrenia.
Atherosclerosis is accompanied by infiltration of macrophages to the intima of blood vessels. There they engulf oxLDL (oxidized low-density lipoproteins) and differentiate to foam cells. These cells are known as major promoters of atherosclerosis progression. In initial experiments I could demonstrate that foam cell formation caused a severe loss in the ability to produce IFNA (interferon A) in response to stimulation with the bacterial cell wall component LPS (lipopolysaccharide). Since IFNA is discussed to have anti-atherosclerotic potential and has the capability to induce immune tolerance, its inhibition in foam cells might promote the atherosclerotic process. For this reason the aim of my PhD project was to clarify the underlying molecular mechanisms that attenuate LPS-induced IFNA expression in foam cells. LPS activates TLR4 (Toll-like receptor 4) in macrophages. Downstream this receptor two distinct signaling pathways are activated, namely a MyD88 (myeloid differentiation primary response gene 88)-dependent and a TRIF (TIR-domain-containing adapter-inducing IFNA)-dependent one. Foam cell formation targeted the TRIF-dependent TLR4 signaling pathway, as seen by loss of IRF3 activation and IFNA expression inhibition, whereas MyD88-initiated NFBB (nuclear factor 'B-light-chain-enhancer' of activated B-cells) activation and subsequent TNF@ (tumor necrosis factor @) expression remained unaltered. The TRIF signaling cascade results in transactivation of the transcription factor IRF3 (interferon regulatory factor 3), the main activator of IFNA expression. This event demands IRF3 phosphorylation by TBK1 (TANK-binding kinase 1), whereas TBK1 needs to be recruited to TRAF3 (TNF receptor associated factor 3) by the scaffold protein TANK (TRAF family member-associated NFBB activator) for its activation. This work allowed to propose the following scheme: OxLDL utilizes SR-A1 (scavenger receptor A1) to activate IRAK4 (interleukin-1 receptor-associated kinase 4), IRAK1 and Pellino3. Active IRAK1 and Pellino3 associate with TRAF3 and Pellino3 promotes mono-ubiquitination of the adaptor molecule TANK. Mono-ubiquitination of TANK interrupts TBK1 recruitment to TRAF3 and thereby abrogates phosphorylation and transactivation of IRF3 as well as subsequent expression of IFNA. In this study I provide evidence for a negative regulatory role of Pellino3 for TRIF-dependent TLR4 signaling. This expands the current knowledge of the interplay between pathways downstream scavenger and Toll-like receptors. Due to the multifaceted roles of TLR4 signaling in pathology, the new TRIF-signaling inhibitor Pellino3 might be of importance as therapeutical target for disease intervention.