PMA and crystal‐induced neutrophil extracellular trap formation involves RIPK1‐RIPK3‐MLKL signaling

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside‐in signaling pathway triggering NET formation is unknown. Here, we show that the receptor‐interacting protein kinase (RIPK)‐1‐stabilizers necrostatin‐1 or necrostatin‐1s and the mixed lineage kinase domain‐like (MLKL)‐inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal‐ or PMA‐induced NET formation in human and mouse neutrophils. These compounds do not affect PMA‐ or urate crystal‐induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA‐induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal‐induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.     

Mechanism of misfolding of the human prion protein revealed by a pathological mutation

The misfolding and aggregation of the human prion protein (PrP) is associated with transmissible spongiform encephalopathies (TSEs). Intermediate conformations forming during the conversion of the cellular form of PrP into its pathological scrapie conformation are key drivers of the misfolding process. Here, we analyzed the properties of the C-terminal domain of the human PrP (huPrP) and its T183A variant, which is associated with familial forms of TSEs. We show that the mutation significantly enhances the aggregation propensity of huPrP, such as to uniquely induce amyloid formation under physiological conditions by the sole C-terminal domain of the protein. Using NMR spectroscopy, biophysics, and metadynamics simulations, we identified the structural characteristics of the misfolded intermediate promoting the aggregation of T183A huPrP and the nature of the interactions that prevent this species to be populated in the wild-type protein. In support of these conclusions, POM antibodies targeting the regions that promote PrP misfolding were shown to potently suppress the aggregation of this amyloidogenic mutant.

The misfolding and aggregation of the human prion protein (PrP) is associated with a number of fatal neurodegenerative disorders designated as transmissible spongiform encephalopathies (TSEs), including Kuru, Creutzfeldt–Jakob disease, fatal familial insomnia, and mad cow disease (1). In its physiological form, the cellular PrP (PrP^C) is a 23-kDa monomeric glycosylated protein that is linked to the outer surface of the neuronal plasma membrane via a glycophosphatidylinositol (GPI) anchor (2). Under conditions associated with TSEs, PrP^C misfolds into a nonnative conformation (scrapie PrP^Sc) that is prone to aggregation into insoluble amyloid fibrils (3). The conversion into PrP^Sc has been proposed to be able to propagate from one cell to another (4). Genetic traits also exist linking point mutations of PrP and familial forms of TSEs. These pathological mutations are mostly located in the C-terminal globular domain of the PrP^C and have been shown to generally enhance the aggregation propensity of PrP (5, 6).While the pathological relevance of PrP is now established, its function remains highly debated. PrP^C has been shown to be involved in the maintenance of myelin on peripheral nerves (7), and evidence exists for a number of other putative physiological roles, including calcium modulation, copper sensing, long-term potentiation, and long-term memory (8). A well-characterized property of the physiological PrP^C is its native structure, which is composed of a disordered N-terminal flexible tail (residues 23–124) and a structured C-terminal region (125–230) composed of three α-helices and a short antiparallel β-sheet (9–11). Recently, structures of amyloid states of PrP generated in vitro have been resolved, providing insights about the possible in vivo form of PrP^Sc (12, 13). Despite the progress made in the structural characterization of PrP^Sc, there are major gaps in our understanding of the mechanisms that trigger the misfolding and aggregation of PrP^C under conditions associated with the insurgence and development of TSEs. A major challenge in this context is the study of metastable misfolding intermediates, which are elusive to conventional experimental techniques for structure determination because of their transient and heterogeneous nature (14). It is now generally acknowledged that the initial step for the misfolding of the C-terminal domain of PrP^C is the disruption of the native packing of the region formed by the two β-strands S1 and S2 and the α-helix H1, against the region composed of the helices H2–H3 (15). The detachment of these two subdomains, denoted as S1–H1–S2 and H2–H3, respectively, is prevented under native conditions by four main intramolecular “gatekeeper” interactions (D178–R164, T183–Y162, H187–R156, D202–R156). Each of these interactions is altered by specific pathological mutations associated with inherited forms of TSEs (D178N, T183A, H187R, D202N), suggesting a destabilization of the native interface between the two subdomains (15). Among these PrP variants, a mutation associated with very early-onset dementia and spongiform encephalopathy in patients, namely T183A (16), abolishes a crucial hydrogen bond between Y162 from the β-strand S2 and T183 from the α-helix H3. Under physiological conditions, this H-bond stabilizes the packing at the interface between the subdomains S1–H1–S2 and H2–H3 (SI Appendix, Fig. S1) (17), and its depletion by T183A induces the strongest destabilization of the PrP^C structure among the TSE-associated PrP variants (18).We addressed in the present study the fundamental early molecular mechanism of human PrP^C (huPrP^C) misfolding induced by T183A using NMR experiments in combination with biophysical investigations and enhanced molecular metadynamics simulations. The study compared the properties of wild-type (WT) and T183A huPrP^C and identified a misfolded intermediate species that acts as a precursor to the formation of amyloid aggregates by the C-terminal domain of the mutant (residues 125–230) despite the fact that this construct lacks the amyloidogenic region 106–126 (19). We provide conclusive evidence of this aggregation mechanism using POM antibodies (Abs) targeting the specific epitope that was here found to initiate the misfolding of T183A huPrP^C. Taken together, these results generate a new detailed understanding of the structural transitions in huPrP^C that trigger its amyloid formation and provide proof of principle for a structure-based identification of targeted molecular strategies to prevent huPrP^C misfolding and aggregation.     

A new incision design for mandibular symphysis bone-grafting procedures

BACKGROUND: Traditional methods of procuring mandibular symphysis bone grafts may leave soft tissue scarring, and cause paresthesia and lip droop. METHODS: Nineteen patients selected for treatment were given general health, periodontal, and radiographic evaluations. Patients had inadequate bone volume for dental implant placement or required preprosthetic ridge augmentation procedures. Prior to surgery, bone sounding was performed to determine tissue thickness. All patients had a minimum of 4 mm of keratinized gingiva. Under local anesthesia, incisions were initiated within the keratinized gingiva. Full-thickness mucoperiosteal flaps were elevated, and small burs were used to obtain bone blocks from the mandibular symphysis. A bone-scraping device was used to obtain strips of cortical bone. A combination of sling and interrupted sutures was used for wound closure. RESULTS: All patients healed uneventfully without wound dehiscence, paresthesia, or lip droop. Sufficient bone was obtained for ridge or sinus augmentation with eventual implant placement. CONCLUSIONS: A new incision design is presented. This flap design is carried out within keratinized gingiva. Limiting the flap design to keratinized tissue facilitates flap closure and avoids wound dehiscence.     

Effects of melatonin in connection with the antioxidant defense system in the gills of the estuarine crab Neohelice granulata

Numerous studies have shown that melatonin exerts some influence on the antioxidant defense system (ADS) in vertebrates, but for crustaceans no such effect has been demonstrated till now. However, earlier reports did show a similar profile of daily variations in the ADS of the gills and the melatonin content of the eyestalk in the crab Neohelice granulata and, thus, the aim of this study was to take a closer look at the effects of melatonin in the gill ADS of N. granulata. Gill ADS is to a minor extent modulated by reactive oxygen species (ROS), because only the nonproteic sulfhydryl (NP-SH) content increases (p < 0.05) in the presence of hydrogen peroxide (H₂O₂). No significant differences (p > 0.05) were observed in the melatonin content of the hemolymph between intact and eyestalkless crabs. Gills from intact and eyestalkless crabs injected with physiological saline showed a daily variation in the total peroxyl radical scavenging capacity (TPRSC) (p < 0.05) with two peaks, one at the photophase and another at the scotophase. However, in the gills of eyestalkless crabs injected with melatonin (2 × 10⁻¹² mol crab⁻¹), the daily variation in TPRSC values was abolished (p > 0.05). This molecule did not change the NP-SH content (p > 0.05) in vitro, but decreased (p < 0.05) the oxygen consumption in gills when incubated for 120 min. In the in vivo experiments melatonin also decreased (p < 0.05) the oxygen consumption in eyestalkless crabs after 390 min. The results suggest that melatonin does not act directly on the ADS of the gills of N. granulata, but decreases the aerobic metabolism possibly involved in variations of tissue ADS.     

Reduced hemodynamic load and cardiac hypotrophy in patients with anorexia nervosa

BACKGROUND: Anorexia nervosa is associated with lower left ventricular mass (LVM) and systolic dysfunction. Whether these abnormalities reflect chronic protein-energy malnutrition or are primarily related to lower cardiac workload is unclear. OBJECTIVE: The objective of the study was to verify whether low LVM in anorexia nervosa is explained by low hemodynamic load. DESIGN: Ninety-one women with anorexia nervosa [macro x +/- SD age: 20.5 +/- 6.1 y; body mass index (in kg/m(2)): 15.6 +/- 1.9; group 1] and 62 normal-weight female control subjects (age: 22.5 +/- 5.5 y; body mass index: 20.9 +/- 1.2; group 2) underwent Doppler echocardiography. LVM was evaluated as the percentage predicted by body height, sex, and stroke work (systolic blood pressure x stroke volume). RESULTS: The left ventricular chamber dimension was smaller and the chamber walls were thinner in group 1 than in group 2, which resulted in significantly lower LVM and LVM indexes (P < 0.0001). Ejection fraction, heart rate, stroke volume, and cardiac output were significantly (P < 0.007) lower in group 1, but peripheral resistance was substantially higher (P < 0.0001). The deviation of LVM from predicted values was lower and the proportion of subjects with inadequate LVM was significantly higher in group 1 than in group 2 (P < 0.0001). This difference was attenuated after adjustment for body weight and heart rate. There were no relations between LVM and laboratory tests in group 1. CONCLUSIONS: Anorexia nervosa is a condition of low hemodynamic load that leads to low LVM. Even with adjustment for stroke work, however, LVM is lower than would be predicted by height, because of the effect of body weight reduction (ie, wasting of lean body mass).     

Australian drinkers’ perceptions of alcohol-related risk by consumption status

Background: This study investigated Australian drinkers’ alcohol-related beliefs according to their alcohol risk status. The primary aims were to assess drinkers’ awareness of the association between alcohol consumption and a range of health consequences and their understanding of the degree of risk represented by their own alcohol consumption.

Method: An online survey was administered to 2168 drinkers who consume alcohol at least twice per month. Respondents reported their alcohol intake levels and their beliefs relating to the relationship between alcohol and shorter-term (proximal) risks (e.g., drink-driving) and longer-term (distal) risks (e.g., stroke and cancer).

Results: Just over half (52%) of those drinking at high or very high risk levels did not perceive their drinking to be harmful. A large majority (85%) of the sample was aware of various short-term risks of excessive alcohol consumption, but only half appeared aware of the association between alcohol consumption and more distal health conditions.

Conclusions: The relatively low levels of awareness of the alcohol–disease link and the weak relationship between perceived risk and alcohol consumption levels suggest that attempts to reduce current high levels of alcohol-related harm could include public education campaigns designed to (i) improve drinkers’ understanding of the prevalence of alcohol-related harms upon which current alcohol guidelines are based, (ii) prompt drinkers to review their intake levels in the light of the guidelines to assess their potential risk of harm, and (iii) make alcohol-related risks more salient to every-day consumption decisions.     

Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium

A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.     

General joint frailty model for recurrent event data with a dependent terminal event: Application to follicular lymphoma data

Many biomedical studies focus on delaying disease relapses and on prolonging survival. Usual methods only consider one event, often the first recurrence or death. However, ignoring the other recurrences may lead to biased results. The whole history of the disease should be considered for each patient. In addition, some diseases involve recurrences that can increase the risk of death. In this case, the death time may be dependent on the recurrent event history. We propose a joint frailty model to analyze recurrences and death simultaneously. Two gamma-distributed frailties take into account both the inter-recurrences dependence and the dependence between the recurrences and the survival times. We estimate separate parameters for disease recurrent event times and survival times in the joint frailty model to distinguish treatment effects and prognostic factors on these two types of events. We show how maximum penalized likelihood estimation can be applied to semiparametric estimation of the continuous hazard functions in the proposed joint frailty model with right censoring. We also propose parametrical approach. We evaluate the model by simulation studies and illustrate through a study of patients with follicular lymphoma.     

Antioxidant and free-radical scavenging activity of constituents of the leaves of Tachigalia paniculata

Two new myricetin glycosides, myricetin 7-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (1) and myricetin 7-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (2), together with the known compounds quercetin 3-O-beta-D-glucopyranoside (3), quercetin 3-O-alpha-L-rhamnopyranoside (4), quercetin 3-O-beta-D-galactopyranoside (5), methyl gallate (6), isovanillin (7), 4-hydroxymethylbenzoate (8), 3,4-dihydroxymethylbenzoate (9), and caffeoyl aldehyde (10) were isolated from the leaves of Tachigalia paniculata. The structures of these compounds were determined by spectroscopic methods. Their antioxidant activity was determined by measuring free-radical scavenging effects using three different assays, namely, the Trolox Equivalent Antioxidant Capacity (TEAC) assay, the coupled oxidation of beta-carotene and linoleic acid (autoxidation assay), and the inhibition of xanthine oxidase activity. Compounds 1, 2, and 6 showed activity in the TEAC test, compounds 5-7 and 10 were moderately active in the autoxidation assay, while compounds 1 and 2 were the most potent of the isolates in the xanthine oxidase test.     

Cerebellar tumour presenting with pathological laughter and gelastic syncope

There is no report of patients in whom pathological laughter, a rare condition characterized by uncontrollable episodes of laughter usually triggered by unrelated stimuli, was ever closely associated with a loss of consciousness overtly linked with the onset of such uncontrollable laughter, also referred to as a gelastic syncope. A 53-year-old man presented with a 4-month history of syncope following intense and uncoordinated laughter. Physical and neurological examination was normal and the patient had no other typical cerebellar signs. We found a mass in the cerebellar vermis abutting the floor of the fourth ventricle, which upon histological examination after surgery proved to be an ependymoma. We emphasize that pathological laughter and gelastic syncope could represent unique and sole features of a cerebellar disorder.