A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner.

BACKGROUND: STAT1 is an intracellular signaling molecule that is crucially involved in the regulation of the innate immune system by activation of defense mechanisms against microbial pathogens. Phosphorylation-dependent activation of the STAT1 transcription factor is associated with a conversion from an antiparallel to parallel dimer configuration, which after nuclear import binds to DNA. However, not much is known about the specific intermolecular interactions that stabilize unphosphorylated, antiparallel STAT1 complexes prior to activation. RESULTS: In this study, we identified a previously unknown interdimeric interaction site, which is involved in the termination of STAT1 signaling. Introduction of the glutamic acid-to-alanine point mutation E169A in the coiled-coil domain (CCD) by site-directed mutagenesis led to increased tyrosine phosphorylation as well as accelerated and prolonged nuclear accumulation in transiently transfected cells. In addition, DNA-binding affinity and transcriptional activity were strongly enhanced in the substitution mutant compared to the wild-type (WT) protein. Furthermore, we have demonstrated that the E169 residue in the CCD mediates the release of the dimer from the DNA in an auto-inhibitory manner. CONCLUSION: Based on these findings, we propose a novel mechanism for the inactivation of the STAT1 signaling pathway, assigning the interface with the glutamic acid residue 169 in the CCD a crucial role in this process. Video Abstract.

Acute stress - but not aversive scene content - impairs spatial configuration learning.

Contextual learning pervades our perception and cognition and plays a critical role in adjusting to aversive and stressful events. Our ability to memorise spatial context has been studied extensively with the contextual cueing paradigm, in which participants search for targets among simple distractor cues and show search advantages for distractor configurations that repeat across trials. Mixed evidence suggests that confrontation with adversity can enhance as well as impair the contextual cueing effect. We aimed to investigate this relationship more systematically by devising a contextual cueing task that tests spatial configuration learning within complex visual scenes that were emotionally neutral or negative (Study 1) and was preceded by the Maastricht Acute Stress Test (MAST) or a no-stress control condition (Study 2). We demonstrate a robust contextual cueing effect that was comparable across negative and neutral scenes (Study 1). In Study 2, acute stress disrupted spatial configuration learning irrespective of scene valence and endogenous cortisol reactivity to stress. Together with the emerging evidence in the literature, our findings suggest that spatial configuration learning may be subject to complex regulation as a function of spatial or temporal proximity to a stressor, with potential implications for the development of stress-related psychopathology.

[Successful Biocompatible Treatment of Diaphragmatic Hernia in a Rat Model]. / Erfolgreiche biokompatible Therapie von Zwerchfelldefekten im Rattenmodell.

Treatment of major congenital diaphragmatic hernia (CDH) is a challenging task in paediatric surgery. Gore-Tex® is now commonly used to treat CDH, but it carries the risk of recurrences, infections and other complications. The aim of our study was to analyse to what extent Lyoplant® - an acellular, avascular biocompatible collagen mesh - is suitable for CDH in the rat model. METHODS: A median laparotomy was performed in young Wistar Furth rats with a body weight of 155 - 205 g. A defect was created by excising a 1.0 × 1.0 cm muscular segment of the left diaphragm, which was closed by implanting a PTFE mesh (n = 5), or a Lyoplant mesh (n = 6). For control purposes (sham group, n = 2), the defect was closed directly. Each rat was examined frequently for the duration of 12 weeks. After this period, the abdomen was reopened, examined for adhesions and the left diaphragm was explanted for histological examination. RESULTS: All operated Wistar Furth rats exhibited a physiological body weight gain after the procedure. During the above period, no recurrence of CDH could be found, either radiologically or clinically. In all animals (PTFE vs. Lyoplant vs. sham group), strong adhesions of the left liver lobe to the implanted material were found. In contrast to the PTFE mesh, constant tissue remodeling and continuous neovascularisation were found in the Lyoplant group. CONCLUSION: Our results show that Lyoplant can successfully be used for the biocompatible therapy of CDH in the rat model. The extent to which it can also be used to treat congenital diaphragmatic defects must be demonstrated by further experimental and clinical studies.

Hot-spot KIF5A mutations cause familial ALS.

Heterozygous missense mutations in the N-terminal motor or coiled-coil domains of the kinesin family member 5A (KIF5A) gene cause monogenic spastic paraplegia (HSP10) and Charcot-Marie-Tooth disease type 2 (CMT2). Moreover, heterozygous de novo frame-shift mutations in the C-terminal domain of KIF5A are associated with neonatal intractable myoclonus, a neurodevelopmental syndrome. These findings, together with the observation that many of the disease genes associated with amyotrophic lateral sclerosis disrupt cytoskeletal function and intracellular transport, led us to hypothesize that mutations in KIF5A are also a cause of amyotrophic lateral sclerosis. Using whole exome sequencing followed by rare variant analysis of 426 patients with familial amyotrophic lateral sclerosis and 6137 control subjects, we detected an enrichment of KIF5A splice-site mutations in amyotrophic lateral sclerosis (2/426 compared to 0/6137 in controls; P = 4.2 × 10-3), both located in a hot-spot in the C-terminus of the protein and predicted to affect splicing exon 27. We additionally show co-segregation with amyotrophic lateral sclerosis of two canonical splice-site mutations in two families. Investigation of lymphoblast cell lines from patients with KIF5A splice-site mutations revealed the loss of mutant RNA expression and suggested haploinsufficiency as the most probable underlying molecular mechanism. Furthermore, mRNA sequencing of a rare non-synonymous missense mutation (predicting p.Arg1007Gly) located in the C-terminus of the protein shortly upstream of the splice donor of exon 27 revealed defective KIF5A pre-mRNA splicing in respective patient-derived cell lines owing to abrogation of the donor site. Finally, the non-synonymous single nucleotide variant rs113247976 (minor allele frequency = 1.00% in controls, n = 6137), also located in the C-terminal region [p.(Pro986Leu) in exon 26], was significantly enriched in familial amyotrophic lateral sclerosis patients (minor allele frequency = 3.40%; P = 1.28 × 10-7). Our study demonstrates that mutations located specifically in a C-terminal hotspot of KIF5A can cause a classical amyotrophic lateral sclerosis phenotype, and underline the involvement of intracellular transport processes in amyotrophic lateral sclerosis pathogenesis.

Pediatric Maxillary Osteomyelitis: A Case Report of a Rare Entity.

Maxillary osteomyelitis is a rare disease, especially in the pediatric population. We present a case of maxillary osteomyelitis in an eight-year-old girl with favorable outcome. Diagnosis was based on magnetic resonance imaging as well as on direct inspection intra operatively. Treatment should be based primarily on clinical signs (e.g. loose teeth). Teeth should not been extracted if healthy.

Sensitization of ultra-long-range excited-state electron transfer by energy transfer in a polymerized film.

Distance-dependent energy transfer occurs from the Metal-to-Ligand Charge Transfer (MLCT) excited state Ru(bpy)3(2+*) to an anthracene-acrylate derivative (Acr-An) incorporated into the polymer network of a semirigid poly(ethyleneglycol)dimethacrylate monolith. Following excitation, Ru(bpy)3(2+*) to Acr-An triplet energy transfer occurs followed by long-range, Acr-(3)An-Acr-An → Acr-An-Acr-(3)An, energy migration. With methyl viologen dication (MV(2+)) added as a trap, Acr-(3)An + MV(2+) → Acr-An(+) + MV(+) electron transfer results in sensitized electron transfer quenching over a distance of approximately 90 Å.

Structure and dynamics of molecular rods in membranes: application of a spin-labeled rod.

Molecular rods consisting of a hydrophobic backbone and terminally varying functional groups have been synthesized for applications for the functionalization of membranes. In the present study, we employ a spin-labeled analogue of a recently described new class of molecular rods to characterize their dynamic interactions with membranes. By using the different approaches of ESR and NMR spectroscopy, we show that the spin moiety of the membrane-embedded spin-labeled rod is localized in the upper chain/glycerol region of membranes of different compositions. The rod is embedded within the membrane in a tilted orientation to adjust for the varying hydrophobic thicknesses of these bilayers. This orientation does not perturb the membrane structure. The water solubility of the rod is increased significantly in the presence of certain cyclodextrins. These cyclodextrins also allow the rods to be extracted from the membrane and incorporated into preformed membranes. The latter will improve the future applications of these rods in cellular systems as stable membrane-associated anchors for the functionalization of membrane surfaces.

Atom transfer radical polymerization preparation and photophysical properties of polypyridylruthenium derivatized polystyrenes.

A ruthenium containing polymer featuring a short carbonyl-amino-methylene linker has been prepared by atom transfer radical polymerization (ATRP). The polymer was derived from ATRP of the N-hydroxysuccinimide (NHS) derivative of p-vinylbenzoic acid, followed by an amide coupling reaction of the NHS-polystyrene with Ru(II) complexes derivatized with aminomethyl groups (i.e., [Ru(bpy)2(CH3-bpy-CH2NH2)](2+) where bpy is 2,2'-bipyridine, and CH3-bpy-CH2NH2 is 4-methyl-4'-aminomethyl-2,2'-bipyridine). The Ru-functionalized polymer structure was confirmed by using nuclear magnetic resonance and infrared spectroscopy, and the results suggest that a high loading ratio of polypyridylruthenium chromophores on the polystyrene backbone was achieved. The photophysical properties of the polymer were characterized in solution and in rigid ethylene glycol glasses. In solution, emission quantum yield and lifetime studies reveal that the polymer's metal-to-ligand charge transfer (MLCT) excited states are quenched relative to a model Ru complex chromophore. In rigid media, the MLCT-ground state band gap and lifetime are both increased relative to solution with time-resolved emission measurements revealing fast energy transfer hopping within the polymer. Molecular dynamics studies of the polymer synthesized here as well as similar model systems with various spatial arrangements of the pendant Ru complex chromophores suggest that the carbonyl-amino-methylene linker probed in our target polymer provides shorter Ru-Ru nearest-neighbor distances leading to an increased Ru*-Ru energy hopping rate, compared to those with longer linkers in counterpart polymers.

Fluoropolymer-Stabilized Chromophore-Catalyst Assemblies in Aqueous Buffer Solutions for Water-Oxidation Catalysis.

Here, the application of the fluorinated polymer [Dupont AF, a copolymer of 4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole and tetrafluoroethylene] is described in stabilizing phosphonate-derivatized molecular assemblies on oxide electrodes. In the procedure, the polymer was dip-coated onto the surfaces of oxide electrodes with pre-bound, phosphonate-derivatized chromophores and assemblies, including assemblies for water oxidation. The results of the experiments showed a high degree of stabilization by the added polymer and a demonstration of its use in stabilizing surface-bound assemblies for water-oxidation catalysis.

It takes more than a coating to get nanoparticles through the intestinal barrier in vitro.

Size and shape are crucial parameters which have impact on the potential of nanoparticles to penetrate cell membranes and epithelial barriers. Current research in nanotoxicology additionally focuses on particle coating. To distinguish between core- and coating-related effects in nanoparticle uptake and translocation, two nanoparticles equal in size, coating and charge but different in core material were investigated. Silver and iron oxide nanoparticles coated with poly (acrylic acid) were chosen and extensively characterized by small-angle x-ray scattering, nanoparticle tracing analysis and transmission electron microscopy (TEM). Uptake and transport were studied in the intestinal Caco-2 model in a Transwell system with subsequent elemental analysis. TEM and ion beam microscopy were conducted for particle visualization. Although equal in size, charge and coating, the behavior of the two particles in Caco-2 cells was different: while the internalized amount was comparable, only iron oxide nanoparticles additionally passed the epithelium. Our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. Knowledge about the different roles of the particle coating and core materials in crossing biological barriers will facilitate toxicological risk assessment of nanoparticles and contribute to the optimization of pharmacokinetic properties of nano-scaled pharmaceuticals.

Successful treatment of basal cell carcinomas in a nevoid basal cell carcinoma syndrome with topical 5% imiquimod.

Gorlin-Goltz syndrome, also referred to as naevoid basal cell carcinoma syndrome (NBCCS), is an autosomal dominant skin disease with complete penetrance and inconstancy of the four major findings: multiple naevoid basal cell carcinomas (BCCs), pits on palms and soles, skeletal abnormalities (for example, jaw cysts), and ectopic calcification. The treatment of multiple BCCs is still a matter of debate. We report three cases of multiple BCCs in Gorlin-Goltz syndrome treated with topical 5% imiquimod cream, an immune response modifier. Patients were successfully cleared of BCCs after treatment for 6-8 weeks. Histologically no apparent signs of BCC-persistence could be detected and no recurrences were detected during the 12 month follow up period.

Membrane properties of cholesterol analogs with an unbranched aliphatic side chain.

The interactions between cholesterol and other membrane molecules determine important membrane properties. It was shown that even small changes in the molecular structure of cholesterol have a crucial influence on these interactions. We recently reported that in addition to alterations in the tetracyclic ring structure, the iso-branched side chain of cholesterol also has a significant impact on membrane properties (Scheidt et al., 2013). Here we used synthetic cholesterol analogs to investigate the influence of an unbranched aliphatic side chain of different length. The (2)H NMR order parameter of the phospholipid chains and therefore the molecular packing of the phospholipid molecules shows a significant dependence on the sterol's alkyl side chain length, while, membrane permeation studied by a dithionite ion permeation assay and lateral diffusion measured by (1)H MAS pulsed field gradient NMR are less influenced. To achieve the same molecular packing effect similar to that of an iso-branched aliphatic side chain, a longer unbranched side chain (n-dodecyl instead of n-octyl) at C17 of cholesterol is required. Obviously, sterols having a branched iso-alkyl chain with two terminal methyl groups exhibit altered cholesterol-phospholipid interactions compared to analogous molecules with a straight unbranched chain.

Time dependent effects of stress prior to encoding on event-related potentials and 24 h delayed retrieval.

Stress can exert profound effects on memory encoding. Here, we investigated whether (sub)cortical information processing during encoding and memory retrieval at a 24 h delayed test are affected by the temporal proximity between stress and memory encoding. Sixty-four participants engaged in the Maastricht Acute Stress Test (MAST) or a no-stress control condition either immediately before (i.e., proximate condition) or 30 min before (i.e., distant condition) a picture encoding task. In general, stress decreased the number of freely recalled and recognized pictures and increased the number of false alarms. However, timing of stress exposure did not differentially affect picture recall, recognition or selective attention processes (i.e., LPP). Nevertheless, stress-induced cortisol responses and correctly recognized neutral pictures were positively associated within the proximate stress condition but negatively associated within the distant stress condition. These findings suggest that the time at which a stressor is applied might differentially impact the association between stress-induced cortisol elevations and memory formation and indicate the need for a finer delineation of the time window during which glucocorticoids affect memory formation processes.

Introducing the Maastricht Acute Stress Test (MAST): a quick and non-invasive approach to elicit robust autonomic and glucocorticoid stress responses.

Stress-related research has employed several procedures to activate the human stress system. Two of the most commonly used laboratory paradigms are the Trier Social Stress Test (TSST) and the Cold Pressor Test (CPT). We combined their most stressful features to create a simple laboratory stress test capable of eliciting strong autonomic and glucocorticoid stress responses. In comparison with the CPT and its variations, our stress tool (labeled the Maastricht Acute Stress Test; MAST) was found to yield superior salivary cortisol responses, while being equally effective in eliciting subjective stress reactions and (systolic and diastolic) blood pressure increases (study 1; N=80). In study 2 (N=20), we directly compared the effectiveness of the MAST and TSST and found that both methods elicited similar subjective, salivary alpha-amylase, and salivary cortisol stress responses. Finally, we developed and evaluated an appropriate no-stress control version of the MAST that was similar to the stress version, although it did not comprise stressful components (study 3; N=40). Collectively, our results confirm the effectiveness of the MAST in terms of subjective, autonomic, and--most importantly--glucocorticoid stress responses. Thus, as a brief and simple stress protocol, the MAST holds considerable promise for future research.

Xenogeneic islet transplantation of microencapsulated porcine islets for therapy of type I diabetes: long-term normoglycemia in STZ-diabetic rats without immunosuppression.

PURPOSE: Rejection and possible infection with porcine pathogens are obstacles in clinical xenogeneic transplantation of porcine pancreatic islets (PPI) to treat diabetic patients. A solution to this problem could be microencapsulation of the PPI. However, isolation and microencapsulation are highly demanding tasks with considerable risks of damaging the PPI. Thus, it is not surprising that the long-term function (>200 days) of microencapsulated PPI (mPPI), transplanted to diabetic rats, has been observed only in a few cases. METHODS: Diabetes was induced in Wistar rats with streptozotozin (STZ 60 mg/kg body weight). Animals with consecutive blood glucose levels >300 mg/dl for more than 2 days were considered diabetic. PPI were isolated from brain-dead hybrid pigs (age 6-7 months or 2-3 years) using the Ricordi-technique and Liberase(PI). After in vitro culture PPI were microencapsulated with highly purified barium-alginate and 1,000 mPPI of 300-500 microm Ø were transplanted under the left kidney capsule and/or into the peritoneal cavity of STZ-diabetic rats (n = 15) without immunosuppression. Daily, later weekly, blood glucose level and body-weight were measured. RESULTS: mPPI showed normal glucose tolerance in vitro and also in vivo. Normoglycemia occurred between day 1 and 15 after transplantation. Four mPPI grafts functioned for more than 230 days, the longest now for >550 days. Three rats are currently normoglycemic for >40 days. Six rats lost xenograft function after 12-20 days, due to inflammatory reactions at the site of the grafts. Two xenografts failed to induce normoglycemia, because the capsules did not contain enough viable PPI. CONCLUSIONS: Microencapsulated xenogeneic islets can induce long term normoglycemia in rats without immunosuppression. However, very often the grafts fail to control the blood glucose level adequately. The reasons for these failures are currently under investigation. Nevertheless, our results are very promising and might lead the way towards preclinical trials in non-human primates.

Severe Guillain-Barré syndrome associated with chromosome 17p11.2-12 duplication.

We report a patient with Guillain-Barré syndrome (GBS), characterized by severe tetraparesis, bulbar syndrome, and ophthalmoparesis. The nadir was reached within 1 day, followed by respiratory insufficiency requiring mechanical ventilation. Molecular analysis revealed a duplication at chromosome 17p11.2-12, which is a known genetic cause of Charcot-Marie-Tooth disease type 1A (CMT1A). We suggest that this genotype may comprise a previously unrecognized genetic risk factor for GBS.

A new biocompatible material (Lyoplant) for the therapy of congenital abdominal wall defects: first experimental results in rats.

Congenital abdominal wall defects are impressive and dramatic malformations. Common surgical therapy for omphalocele and gastroschisis is to place the herniated viscera back into the abdomen and to close the fascia. Small defects can be closed directly by surgical treatment. In large defects, resorbable and non-resorbable artificial materials are necessary to close the fascia. The aim of this study is to find out whether new biocompatible materials might be suitable for the treatment of such abdominal wall defects. A median laparotomy was performed in young Wistar rats with a body weight of 75-100 g. Then a full thickness defect was created by excising a 1.5 x 2.5 cm segment including fascia, muscles and peritoneum. These defects were then closed by implantation of a PTFE mesh (Dual-Mesh, n = 6), a PPP mesh (Prolene, n = 6) or a new biocompatible mesh (NBM; Lyoplant, n = 6). Each rat was examined daily after treatment. Bodyweight was determined and the possible development of a hernia was monitored. After 6 weeks, the abdomen was opened again. Adhesions to the intestine were measured and the abdominal wall was removed for histological and tensiometric examination. (1) Compared to the untreated controls, all animals showed physiologic growth and normal bodyweight curve. (2) Only in one rat (Prolene) did an abdominal hernia develop. (3) In contrast to PTFE and PPP mesh, NBM showed only minimal adhesion to the intestine. (4) Tensiometry revealed high stability for non-resorbable materials. However, the characteristics of NBM were very similar to untreated abdominal wall. Our initial results indicate that biocompatible materials can also be used for the therapy of congenital abdominal wall defects.