Targeting type I DED interactions at the DED filament serves as a sensitive switch for cell fate decisions.

Activation of procaspase-8 in the death effector domain (DED) filaments of the death-inducing signaling complex (DISC) is a key step in apoptosis. In this study, a rationally designed cell-penetrating peptide, DEDid, was engineered to mimic the h2b helical region of procaspase-8-DED2 containing a highly conservative FL motif. Furthermore, mutations were introduced into the DEDid binding site of the procaspase-8 type I interface. Additionally, our data suggest that DEDid targets other type I DED interactions such as those of FADD. Both approaches of blocking type I DED interactions inhibited CD95L-induced DISC assembly, caspase activation and apoptosis. We showed that inhibition of procaspase-8 type I interactions by mutations not only diminished procaspase-8 recruitment to the DISC but also destabilized the FADD core of DED filaments. Taken together, this study offers insights to develop strategies to target DED proteins, which may be considered in diseases associated with cell death and inflammation.

Spinal cord gray matter atrophy is associated with functional decline in post-polio syndrome.

OBJECTIVE: To determine if patients with post-polio syndrome (PPS) show spinal cord gray matter (SCGM) atrophy and to assess associations between SCGM atrophy, muscle strength and patient-reported functional decline. METHODS: Twenty patients diagnosed with PPS (March of Dimes criteria) and 20 age- and sex-matched healthy controls (HC) underwent 3T axial 2D-rAMIRA magnetic resonance imaging at the intervertebral disc levels C2/C3-C6/C7, T9/T10 and the lumbar enlargement level (Tmax ) (0.5 × 0.5 mm2 in-plane resolution). SCGM areas were segmented manually by two independent raters. Muscle strength, self-reported fatigue, depression and pain measures were assessed. RESULTS: Post-polio syndrome patients showed significantly and preferentially reduced SCGM areas at C2/C3 (p = 0.048), C3/C4 (p = 0.001), C4/C5 (p < 0.001), C5/C6 (p = 0.004) and Tmax (p = 0.041) compared to HC. SCGM areas were significantly associated with muscle strength in corresponding myotomes even after adjustment for fatigue, pain and depression. SCGM areaTmax together with age and sex explained 68% of ankle dorsiflexion strength variance. No associations were found with age at or time since infection. Patients reporting PPS-related decline in arm function showed significant cervical SCGM atrophy compared to stable patients adjusted for initial disease severity. CONCLUSIONS: Patients with PPS show significant SCGM atrophy that correlates with muscle strength and is associated with PPS-related functional decline. Our findings suggest a secondary neurodegenerative process underlying SCGM atrophy in PPS that is not explained by aging or residua of the initial infection alone. Confirmation by longitudinal studies is needed. The described imaging methodology is promising for developing novel imaging surrogates for SCGM diseases.

Regulation of extrinsic apoptotic signaling by c-FLIP: towards targeting cancer networks.

The extrinsic pathway is mediated by death receptors (DRs), including CD95 (APO-1/Fas) or TRAILR-1/2. Defects in apoptosis regulation lead to cancer and other malignancies. The master regulator of the DR networks is the cellular FLICE inhibitory protein (c-FLIP). In addition to its key role in apoptosis, c-FLIP may exert other cellular functions, including control of necroptosis, pyroptosis, nuclear factor κB (NF-κB) activation, and tumorigenesis. To gain further insight into the molecular mechanisms of c-FLIP action in cancer networks, we focus on the structure, isoforms, interactions, and post-translational modifications of c-FLIP. We also discuss various avenues to target c-FLIP in cancer cells for therapeutic benefit.

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1ß-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Normalization of Spinal Cord Total Cross-Sectional and Gray Matter Areas as Quantified With Radially Sampled Averaged Magnetization Inversion Recovery Acquisitions.

Background: MR imaging of the spinal cord (SC) gray matter (GM) at the cervical and lumbar enlargements' level may be particularly informative in lower motor neuron disorders, e. g., spinal muscular atrophy, but also in other neurodegenerative or autoimmune diseases affecting the SC. Radially sampled averaged magnetization inversion recovery acquisition (rAMIRA) is a novel approach to perform SC imaging in clinical settings with favorable contrast and is well-suited for SC GM quantitation. However, before applying rAMIRA in clinical studies, it is important to understand (i) the sources of inter-subject variability of total SC cross-sectional areas (TCA) and GM area (GMA) measurements in healthy subjects and (ii) their relation to age and sex to facilitate the detection of pathology-associated changes. In this study, we aimed to develop normalization strategies for rAMIRA-derived SC metrics using skull and spine-based metrics to reduce anatomical variability. Methods: Sixty-one healthy subjects (age range 11-93 years, 37.7% women) were investigated with axial two-dimensional rAMIRA imaging at 3T MRI. Cervical and thoracic levels including the level of the cervical (C4/C5) and lumbar enlargements (Tmax) were examined. SC T2-weighted sagittal images and high-resolution 3D whole-brain T1-weighted images were acquired. TCA and GMAs were quantified. Anatomical variables with associations of |r| > 0.30 in univariate association with SC areas, and age and sex were used to construct normalization models using backward selection with TCAC4/C5 as outcome. The effect of the normalization was assessed by % relative standard deviation (RSD) reductions. Results: Mean inter-individual variability and the SD of the SC area metrics were considerable: TCAC4/5: 8.1%/9.0; TCATmax: 8.9%/6.5; GMAC4/C5: 8.6%/2.2; GMATmax: 12.2%/3.8. Normalization based on sex, brain WM volume, and spinal canal area resulted in RSD reductions of 23.7% for TCAs and 12.0% for GM areas at C4/C5. Normalizations based on the area of spinal canal alone resulted in RSD reductions of 10.2% for TCAs and 9.6% for GM areas at C4/C5, respectively. Discussion: Anatomic inter-individual variability of SC areas is substantial. This study identified effective normalization models for inter-subject variability reduction in TCA and SC GMA in healthy subjects based on rAMIRA imaging.

Pharmacological targeting of c-FLIPL and Bcl-2 family members promotes apoptosis in CD95L-resistant cells.

The development of efficient combinatorial treatments is one of the key tasks in modern anti-cancer therapies. An apoptotic signal can either be induced by activation of death receptors (DR) (extrinsic pathway) or via the mitochondria (intrinsic pathway). Cancer cells are characterized by deregulation of both pathways. Procaspase-8 activation in extrinsic apoptosis is controlled by c-FLIP proteins. We have recently reported the small molecules FLIPinB/FLIPinBγ targeting c-FLIPL in the caspase-8/c-FLIPL heterodimer. These small molecules enhanced caspase-8 activity in the death-inducing signaling complex (DISC), CD95L/TRAIL-induced caspase-3/7 activation and subsequent apoptosis. In this study to increase the pro-apoptotic effects of FLIPinB/FLIPinBγ and enhance its therapeutic potential we investigated costimulatory effects of FLIPinB/FLIPinBγ in combination with the pharmacological inhibitors of the anti-apoptotic Bcl-2 family members such as ABT-263 and S63845. The combination of these inhibitors together with FLIPinB/FLIPinBγ increased CD95L-induced cell viability loss, caspase activation and apoptosis. Taken together, our study suggests new approaches for the development of combinatorial anti-cancer therapies specifically targeting both intrinsic and extrinsic apoptosis pathways.

Hydroxyethyl starch-induced pruritus: clinical characteristics and influence of dose, molecular weight and substitution.

Severe persistent pruritus is a common, but incompletely characterized, complication of hydroxyethyl starch (HES) infusion. This retrospective study aimed to assess HES-induced pruritus by electron microscopic findings, pruritus characteristics, and response to stimuli, and to determine the impact of HES dosage, molecular weight and substitution. Seventy patients with electron micro-scopy-proven HES-induced pruritus were included. HES-laden vacuoles were observed in skin macrophages of all patients. The median latency between HES exposure and pruritus onset was 3 weeks, and the median duration of pruritus was 6 months. Pruritus was severe, or very severe, in 80% of patients. Mechanical stimuli triggered pruritus in 74% of patients. Although the median cumulative dose of HES was 300 g, 15% of patients developed pruritus after only 30 g. There were no significant differences between HES 130/0.4 and HES 200/0.5 in pruritus latency, duration or severity. HES-induced pruritus thus may occur at any dose, molecular weight or substitution.

Population dynamics among methicillin-resistant Staphylococcus aureus isolates in Germany during a 6-year period.

Methicillin-resistant Staphylococcus aureus (MRSA) originated from the health care setting but is now emerging in communities without health care contact (CA-MRSA) or in livestock (LA-MRSA). The impact on the whole MRSA population was assessed in a German prospective multicenter study. Thirty-three laboratories consecutively collected up to 50 MRSA isolates from infection or carriage during two sampling periods in 2004 to 2005 and 2010 to 2011. Patient-related data were collected using a standardized questionnaire. Methicillin resistance was confirmed by the detection of mecA or its homologue mecA(LGA251). The spa type and major virulence factors were analyzed for each isolate. In total, 1,604 (2004 to 2005) and 1,603 (2010 to 2011) MRSA isolates were analyzed; one isolate from each sampling period harbored mecA(LGA251). LA-MRSA increased significantly (odds ratio [OR] = 22.67, 95% confidence interval [CI] = 8.51 to 85.49, P < 0.0005) and spread over Germany, originating from northwestern regions. Panton-Valentine leukocidin-positive CA-MRSA rose significantly, particularly in southern Germany, but the proportion in 2010 to 2011 remained low (2.7%, OR = 2.80, 95% CI = 1.54 to 5.34, P < 0.0005). The emerging MRSA clones changed the MRSA population in Germany during a 6-year period significantly. The ongoing epidemiological shift and changes of MRSA sources create a need for revision of guidelines for MRSA infection control and treatment.

Retinoic acid induces expression of the thyroid hormone transporter, monocarboxylate transporter 8 (Mct8).

Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. Mct8 (monocarboxylate transporter 8), expressed predominantly in the brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells toward neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All-trans-RA (tRA) and other RA receptor (RAR) agonists dramatically (>300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1- and 4.3-fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and 5'-rapid amplification of cDNA ends PCR analysis in F9 cells identified 11 transcription start sites and a proximal Sp1 site but no TATA box. tRA significantly enhanced Mct8 promoter activity through a consensus RA-responsive element located 6.6 kilobases upstream of the coding region. A chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid X receptor to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates cross-talk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.

Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures.

Induced pluripotent stem cells (iPSCs) outwardly appear to be indistinguishable from embryonic stem cells (ESCs). A study of gene expression profiles of mouse and human ESCs and iPSCs suggests that, while iPSCs are quite similar to their embryonic counterparts, a recurrent gene expression signature appears in iPSCs regardless of their origin or the method by which they were generated. Upon extended culture, hiPSCs adopt a gene expression profile more similar to hESCs; however, they still retain a gene expression signature unique from hESCs that extends to miRNA expression. Genome-wide data suggested that the iPSC signature gene expression differences are due to differential promoter binding by the reprogramming factors. High-resolution array profiling demonstrated that there is no common specific subkaryotypic alteration that is required for reprogramming and that reprogramming does not lead to genomic instability. Together, these data suggest that iPSCs should be considered a unique subtype of pluripotent cell.

Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal cells.

The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study, we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that codifferentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). To better characterize the iPGCs, we performed Real-time polymerase chain reaction, microarray, and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure.

Directed differentiation of human-induced pluripotent stem cells generates active motor neurons.

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases.

Signaling in adult stem cells.

Adult stem cells are set aside during development in order to provide a source for replenishment of tissue over time in response to damage or simply wear and tear. The literature suggests that stem cells can be found in most major organ systems, and that they possess defining characteristics, namely the ability to both self-renew and differentiate down one or more specific lineages. Many groups have sought to define stem cell specific physiology in a molecular fashion by identifying those genes specifically expressed in stem cells. Although these data suggest that there are genes frequently found to be upregulated in stem cells from various tissues, they do not definitively demonstrate that these cells all function similarly. There is also considerable data showing how various signaling pathways influence stem cell growth and differentiation. A review of this literature suggests that many of the well-described pathways affect adult mammalian stem cells from different tissues similarly, and that these effects are sometimes unique to stem cells as opposed to their progeny. In this review we summarize the effects of well-known signaling pathways on several of the most well defined stem cells and argue that the similarity with which unique stem cells from different tissues respond to external stimuli suggests that they share functional mechanisms.

Verbal Paired Associates tests limits on validity and reliability.

The Verbal Paired Associates (VPA) subtest from the Wechsler Memory Scale-III (WMS-III) is one of the most widely used instruments for assessing explicit episodic memory performance. The normative data for the VPA subtest in the WMS-III manual show clear evidence of performance ceiling effects that limit the usefulness of this instrument. For this reason, we developed a new 15-item VPA test and we report normative data obtained from a partially stratified sample of 351 healthy adults between 18 and 91 years of age. Only a small fraction of participants obtained perfect scores on our new Paired Associates test. The results show the expected large age-related decline in memory acquisition, indexed by performance on the first study test trial, together with a much smaller age effect on learning across trials. The results also show that performance on the Paired Associates test is related to education, verbal IQ, and to a lesser extent, participants' sex. We provide various equations for precise predictions of Paired Associates test performance.