An Experimental Platform Generating Simulated Blunt Impacts to the Head Due to Rearward Falls.

Impacts to the back of the head due to rearward falls, also referred to as "backfall" events, represent a common source of TBI for athletes and soldiers. A new experimental apparatus is described for replicating the linear and rotational kinematics of the head during backfall events. An anthropomorphic test device (ATD) with a head-borne sensor suite was configured to fall backwards from a standing height, inducing contact between the rear of the head and a ground surface simulant. A pivoting swing arm and release strap were used to generate consistent and realistic head kinematics. Backfall experiments were performed with the ATD fitted with an American football helmet and the resulting linear and rotational head kinematics, as well as calculated injury metrics, compared favorably with those of football players undergoing similar impacts during games or play reconstructions. This test method complements existing blunt impact helmet performance experiments, such as drop tower and pneumatic ram test methods, which may not be able to fully reproduce head-neck-torso kinematics during a backfall event.

The Tingathe Surge: a multi-strategy approach to accelerate HIV case finding in Malawi.

SETTING: Nineteen health facilities in rural, southeastern Malawi. OBJECTIVE: To describe the implementation and results of a 6-week intervention to accelerate human immunodeficiency virus (HIV) case finding. DESIGN: Six HIV testing strategies were simultaneously implemented. Routinely collected data from Ministry of Health registers were used to determine the number of HIV tests performed and of new cases identified. The weekly averages of the total number of tests and new cases before and during the intervention were compared. Testing by age group and sex was described. The percentage yield of new cases was compared by testing strategy. RESULTS: Of 29 703 HIV tests conducted, 1106 (3.7%) were positive. Of the total number of persons tested, 69.5% were women and 75.5% were aged >15 years. The yield of positive test results was 3.5% among women, 4.3% among men, 4.4% among those aged >15 years and 1.5% among those aged ⩽15 years. The average weekly number of tests increased 106.7% from 3337 to 6896 (P = 0.002). The average weekly number of positive cases identified increased 51.9% from 158 to 240 (P = 0.017). The testing strategy with the highest yield resulted in a 6.0% yield; the lowest was 1.3%. The yield for all strategies, except one, was highest in adult men. CONCLUSION: A multi-strategy approach to HIV testing and counseling can be an effective means of accelerating HIV case finding.

[Development and validation of the Subclinical Stress Symptom Questionnaire SSQ-25]. / Entwicklung und Validierung des Subklinischen Stresssymptom-Questionnaire SSQ-25.

BACKGROUND: Stress symptoms are widespread in the general population and often occur in the early course of mental disorders. However, no validated instrument was available for the study of subclinical stress symptoms and their relevance in the study of psychopathological trajectories. In order to advance and systematize the study of the etiology and pathogenesis of diseases in subclinical populations, the Subclinical Stress Symptom Questionnaire (SSQ-25) was developed in the present study. METHODS: In the course of three online studies, a total of 1174 subjects were recruited. The first study included item selection and the development of the questionnaire based on the analysis of item parameters, reliability, and exploratory factor analysis. To validate the factor structure, confirmatory factor analysis was used. Validation analyses were applied to distinguish the SSQ-25 from three clinical measures: Beck's Anxiety and Depression Inventory (BAI and BDI), and the Posttraumatic Stress Diagnostic Scale (PDS). In the third study the subclinical property of the instrument was investigated. RESULTS: Exploratory and confirmatory factor analyses revealed and confirmed a two-factor model (psychological and physiological stress symptoms). Cronbach's alpha was 0.95. The subclinical property of the SSQ-25 was confirmed by means of item information functions, scatter plots, residuals, and Koenker-Bassett tests as opposed to established clinical measures. DISCUSSION: The SSQ-25 is a comprehensive, reliable, and valid instrument that allows a valid assessment and differentiation of subclinical stress symptoms.

Designing molecular structure to achieve ductile fracture behavior in a stiff and strong 2D polymer, "graphylene".

As the simplest two-dimensional (2D) polymer, graphene has immensely high intrinsic strength and elastic stiffness but has limited toughness due to brittle fracture. We use atomistic simulations to explore a new class of graphene/polyethylene hybrid 2D polymer, "graphylene", that exhibits ductile fracture mechanisms and has a higher fracture toughness and flaw tolerance than graphene. A specific configuration of this 2D polymer hybrid, denoted "GrE-2" for the two-carbon-long ethylene chains connecting benzene rings in the inherent framework, is prioritized for study. MD simulations of crack propagation show that the energy release rate to propagate a crack in GrE-2 is twice that of graphene. We also demonstrate that GrE-2 exhibits delocalized failure and other energy-dissipating fracture mechanisms such as crack branching and bridging. These results demonstrate that 2D polymers can be uniquely tailored to achieve a balance of fracture toughness with mechanical stiffness and strength.

[Importance of the Watzke-Allen test in diagnostics and staging of macular holes]. / Bedeutung des Watzke-Allen-Tests in der Diagnostik und Stadieneinteilung von Makulaforamina.

BACKGROUND: The Watzke-Allen test (WAT) is a simple diagnostic tool designed for the diagnosis of full thickness macular holes (FTMH) but due to the rapid progress of imaging diagnostics it was replaced by spectral domain optical coherence tomography (SD-OCT) of macular pathologies. The aim of this study was to examine if the WAT is able to distinguish between the different FTMH stages. METHODS: In 57 eyes of 57 patients with clinical evidance of FTMH, the WAT was first performed followed by SD-OCT examination and a distinction was made between a negative (slit beam normal) and a positive sign (slit beam modified as groove and hourglass configuration or completely broken). RESULTS: In 49 out of 57 patients the WAT was positive (hourglass 46 patients and broken 3 patients). Based on the SD-OCT results the following diagnoses were made: lamellar macular holes (LMH, 3 patients), vitreomacular traction (VMT, 4 patients), small macular hole (≤ 250 µm, 5 patients), medium sized macular hole (250-400 µm, 11 patients) and large macular hole (≥ 400 µm, 34 patients). In 91 % of the patients with medium and large FTMH, the WAT was positive, whereas the WAT was positive in only 67 % of patients with small FTMH, VMT and LMH. The sensitivity for large and medium FTMH was 93 % but the specificity was only 33 %. CONCLUSION: The WAT was positive in a high percentage of patients with large and medium sized macular holes as well as patients with small macular holes and LMH. The sensitivity of certain indications for treatment was 93 % but the specificity was only 33 %; therefore, the WAT alone is not suitable for a certain preoperative differentiation of macular alterations.

Primary cilia and autophagic dysfunction in Huntington's disease.

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by a single-gene mutation: a CAG expansion in the huntingtin (HTT) gene that results in production of a mutated protein, mutant HTT, with a polyglutamine tail (polyQ-HTT). Although the molecular pathways of polyQ-HTT toxicity are not fully understood, because protein misfolding and aggregation are central features of HD, it has long been suspected that cellular housekeeping processes such as autophagy might be important to disease pathology. Indeed, multiple lines of research have identified abnormal autophagy in HD, characterized generally by increased autophagic induction and inefficient clearance of substrates. To date, the origin of autophagic dysfunction in HD remains unclear and the search for actors involved continues. To that end, recent studies have suggested a bidirectional relationship between autophagy and primary cilia, signaling organelles of most mammalian cells. Interestingly, primary cilia structure is defective in HD, suggesting a potential link between autophagic dysfunction, primary cilia and HD pathogenesis. In addition, because polyQ-HTT also accumulates in primary cilia, the possibility exists that primary cilia might play additional roles in HD: perhaps by disrupting signaling pathways or acting as a reservoir for secretion and propagation of toxic, misfolded polyQ-HTT fragments. Here, we review recent research suggesting potential links between autophagy, primary cilia and HD and speculate on possible pathogenic mechanisms and future directions for the field.

Cerium oxide nanoparticles protect against Aß-induced mitochondrial fragmentation and neuronal cell death.

Evidence indicates that nitrosative stress and mitochondrial dysfunction participate in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Aß) and peroxynitrite induce mitochondrial fragmentation and neuronal cell death by abnormal activation of dynamin-related protein 1 (DRP1), a large GTPase that regulates mitochondrial fission. The exact mechanisms of mitochondrial fragmentation and DRP1 overactivation in AD remain unknown; however, DRP1 serine 616 (S616) phosphorylation is likely involved. Although it is clear that nitrosative stress caused by peroxynitrite has a role in AD, effective antioxidant therapies are lacking. Cerium oxide nanoparticles, or nanoceria, switch between their Ce(3+) and Ce(4+) states and are able to scavenge superoxide anions, hydrogen peroxide and peroxynitrite. Therefore, nanoceria might protect against neurodegeneration. Here we report that nanoceria are internalized by neurons and accumulate at the mitochondrial outer membrane and plasma membrane. Furthermore, nanoceria reduce levels of reactive nitrogen species and protein tyrosine nitration in neurons exposed to peroxynitrite. Importantly, nanoceria reduce endogenous peroxynitrite and Aß-induced mitochondrial fragmentation, DRP1 S616 hyperphosphorylation and neuronal cell death.

Loss of OPA1 disturbs cellular calcium homeostasis and sensitizes for excitotoxicity.

Optic atrophy 1 (OPA1) mutations cause dominant optic atrophy (DOA) with retinal ganglion cell (RGC) and optic nerve degeneration. The mechanism for the selective degeneration of RGCs in DOA remains elusive. To address the mechanism, we reduced OPA1 protein expression in cell lines and RGCs by RNA interference. OPA1 loss results in mitochondrial fragmentation, deficiency in oxidative phosphorylation, decreased ATP levels, decreased mitochondrial Ca(2+) retention capacity, reduced mtDNA copy numbers, and sensitization to apoptotic insults. We demonstrate profound cristae depletion and loss of crista junctions in OPA1 knockdown cells, whereas the remaining crista junctions preserve their normal size. OPA1-depleted cells exhibit decreased agonist-evoked mitochondrial Ca(2+) transients and corresponding reduction of NAD(+) to NADH, but the impairment in NADH oxidation leads to an overall more reduced mitochondrial NADH pool. Although in our model OPA1 loss in RGCs has no apparent impact on mitochondrial morphology, it decreases buffering of cytosolic Ca(2+) and sensitizes RGCs to excitotoxic injury. Exposure to glutamate triggers delayed calcium deregulation (DCD), often in a reversible manner, indicating partial resistance of RGCs to this injury. However, when OPA1 is depleted, DCD becomes irreversible. Thus, our data show that whereas OPA1 is required for mitochondrial fusion, maintenance of crista morphology and oxidative phosphorylation, loss of OPA1 also results in defective Ca(2+) homeostasis.

Subgaleal hemorrhage in a neonate with factor X deficiency following a non-traumatic cesarean section.

This case report describes a term infant born by a non-traumatic, non-instrumented cesarean section that presented with respiratory failure and severe metabolic acidosis secondary to subgaleal hemorrhage (SGH). Further evaluation revealed a functional factor X deficiency that was initially treated with fresh frozen plasma infusions. This report is significant for the occurrence of a SGH in a non-traumatic delivery and emphasizes the importance of obtaining a coagulopathy evaluation in patients with similar presentations. In addition, this case suggests that the mechanism of injury that causes SGH may occur more frequently than previously thought, but does not become clinically significant in patients without an underlying coagulopathy.

Impact of nitric oxide on metabolism in health and age-related disease.

Nitric oxide (NO) serves as a messenger molecule in a variety of physiological systems and also converts into toxic radical species that can damage cells through a process known as nitrosative stress. While the physiological roles of NO in blood vessel dilation, the nervous system and the immune system are well established, recent studies have begun to investigate the role of NO in metabolism and energy expenditure through modulation of mitochondria. NO appears to stimulate mitochondrial biogenesis in certain situations through activation of proteins such as peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1α (PGC1-α). Because of this link between NO and mitochondrial biogenesis, the role of NO in certain aspects of metabolism, including exercise response, obesity, fat cell differentiation and caloric restriction, are the subject of increasing investigation. In addition to its role in mitochondrial biogenesis, NO also stimulates mitochondrial fragmentation, which can be caused by too much mitochondrial fission or inhibition of mitochondrial fusion and can result in bioenergetic failure. While the contribution of NO-mediated mitochondrial fragmentation to neurodegenerative diseases seems clear, the mechanisms by which NO causes fragmentation are uncertain and controversial. In this review, we discuss the role of NO in manipulation of mitochondrial biogenesis and dynamics and how these events contribute to human health- and age-related disease.

Complex II inhibition by 3-NP causes mitochondrial fragmentation and neuronal cell death via an NMDA- and ROS-dependent pathway.

Mitochondrial respiratory complex II inhibition plays a central role in Huntington's disease (HD). Remarkably, 3-NP, a complex II inhibitor, recapitulates HD-like symptoms. Furthermore, decreases in mitochondrial fusion or increases in mitochondrial fission have been implicated in neurodegenerative diseases. However, the relationship between mitochondrial energy defects and mitochondrial dynamics has never been explored in detail. In addition, the mechanism of neuronal cell death by complex II inhibition remains unclear. Here, we tested the temporal and spatial relationship between energy decline, impairment of mitochondrial dynamics, and neuronal cell death in response to 3-NP using quantitative fluorescence time-lapse microscopy and cortical neurons. 3-NP caused an immediate drop in ATP. This event corresponded with a mild rise in reactive oxygen species (ROS), but mitochondrial morphology remained unaltered. Unexpectedly, several hours after this initial phase, a second dramatic rise in ROS occurred, associated with profound mitochondrial fission characterized by the conversion of filamentous to punctate mitochondria and neuronal cell death. Glutamate receptor antagonist AP5 abolishes the second peak in ROS, mitochondrial fission, and cell death. Thus, secondary excitotoxicity, mediated by glutamate receptor activation of the NMDA subtype, and consequent oxidative and nitrosative stress cause mitochondrial fission, rather than energy deficits per se. These results improve our understanding of the cellular mechanisms underlying HD pathogenesis.

Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons.

Mitochondrial dysfunction is an underpinning event in many neurodegenerative disorders. Less clear, however, is how mitochondria become injured during neuronal demise. Nitric oxide (NO) evokes rapid mitochondrial fission in cortical neurons. Interestingly, proapoptotic Bax relocates from the cytoplasm into large foci on mitochondrial scission sites in response to nitrosative stress. Antiapoptotic Bcl-xL does not prevent mitochondrial fission despite its ability to block Bax puncta formation on mitochondria and to mitigate neuronal cell death. Mitofusin 1 (Mfn1) or dominant-negative dynamin-related protein 1(K38A) (Drp1(k38A)) inhibits mitochondrial fission and Bax accumulation on mitochondria induced by exposure to an NO donor. Although NO is known to cause a bioenergetic crisis, lowering ATP by glycolytic or mitochondrial inhibitors neither induces mitochondrial fission nor Bax foci formation on mitochondria. Taken together, these data indicate that the mitochondrial fission machinery acts upstream of the Bcl-2 family of proteins in neurons challenged with nitrosative stress.

Effects of heat shock on the Mre11/Rad50/Nbs1 complex in irradiated or unirradiated cells.

The mechanism by which hyperthermia sensitizes mammalian cells to ionizing radiation remains to be elucidated, but an overwhelming amount of circumstantial evidence suggests that heat radiosensitization might be mediated by inhibition of double-strand break repair, particularly after exposure of irradiated cells to heat treatments in excess of about 43 degrees C. In mammalian cells, double-strand break repair usually occurs via two pathways, non-homologous end-joining and homologous recombination. Several reports suggest a role for non-homologous end-joining in heat radiosensitization, while others implicate homologous recombination as a target. However, cell lines that are compromised in either the non-homologous end-joining or homologous recombination pathway are still capable of being radiosensitized, suggesting that heat affects both pathways. Indeed, several of the proteins involved in one or both of these pathways have been observed to undergo alterations or translocation after unirradiated or irradiated cells are exposed to heat shock. The work summarized in this review implicates proteins of the Mre11/Rad50/Nbs1 complex as targets for heat radiosensitization.

Effect of temperature on the infectivity of metacercariae of Zygocotyle lunata (Digenea: Paramphistomidae).

As a test of the energy limitation hypothesis (ELH), we predicted that temperature would have a significant influence on the infectivity of metacercariae of the digenetic trematode Zygocotyle lunata. Snails infected with Z. lunata were collected from ponds near Crawfordsville, Indiana, isolated at room temperature, and examined for the release of cercariae. Newly encysted metacercariae were collected and incubated 1-30 days at 1 of 5 temperatures (0, 3, 25, 31, 37 C). Twenty-five cysts were fed to each of 5 or 10 mice per treatment group (temperature). At 17 days postinfection, mice were killed and worms were recovered; data were collected on levels of infection in each group and the total body area of each worm. No worms were found in mice fed cysts that had been held at 0 C or 37 C (after 30 days). There were no differences in prevalence, infectivity, or mean intensity among the 3, 25, and 31 C treatments. Infectivity of metacercariae incubated at 37 C for 1 day was significantly greater than in all other treatments, while infectivity of metacercariae in the 37 C/15-day treatment was significantly lower than in all others. Mean body area of worms at 37 C/15 days was significantly greater than at other temperatures, suggesting density-dependent increases in growth. These results, particularly those from the 37 C treatments, are consistent with the ELH; infectivity was lower at high temperatures or when incubated for more time at 1 temperature (fewer energy reserves). It is suggested that microhabitat conditions experienced by metacercariae of Z. lunata could contribute to longer larval life, thus influencing this parasite's temporal dispersal.

Caspase 7 can cleave tumor necrosis factor receptor-I (p60) at a non-consensus motif, in vitro.

Ligand binding to tumor necrosis factor receptor-I (TNFRI) can promote cell survival or activate the apoptotic caspase cascade. Cytoplasmic interaction of TNFRI with TRAF2 and RIP allows for the activation of JNK and NFkappaB pathways. Alternatively, a carboxy terminal death domain protein interaction motif can recruit TRADD, which then recruits FADD/MORT1, and finally procaspase 8. Aggregation of these components form a death inducing signaling complex, leading to the cleavage and activation of caspase 8. We have found that during apoptosis human TNFRI protein is lost in a caspase-dependent manner. The cytoplasmic tail of human TNFRI was found to be susceptible to caspase cleavage but not by caspase 8. Instead, the downstream executioner caspase 7 was the only caspase capable of cleaving TNFRI, in vitro. Identification and characterization of the cleavage site revealed a derivative of the classic EXD motif that incorporates a glutamate (E) in the P1 position. Using several criteria to establish that caspase activity was responsible for cleavage at this site, we confirmed that caspase 7 can cleave at a GELE motif. Mutation of the cleavage site prevented the apoptosis-associated cleavage of TNFRI. This ability of caspase 7 to cleave at a non-EXD or -DXXD motif suggests that the specificity of caspases may be broader than is currently held.

Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release.

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis.

To test the role of ER luminal environment in apoptosis, we generated HeLa cell lines inducible with respect to calreticulin and calnexin and investigated their sensitivity to drug-dependent apoptosis. Overexpression of calreticulin, an ER luminal protein, resulted in an increased sensitivity of the cells to both thapsigargin- and staurosporine-induced apoptosis. This correlated with an increased release of cytochrome c from the mitochondria. Overexpression of calnexin, an integral ER membrane protein, had no significant effect on drug-induced apoptosis. In contrast, calreticulin-deficient cells were significantly resistant to apoptosis and this resistance correlated with a decreased release of cytochrome c from mitochondria and low levels of caspase 3 activity. This work indicates that changes in the lumen of the ER amplify the release of cytochrome c from mitochondria, and increase caspase activity, during drug-induced apoptosis. There may be communication between the ER and mitochondria, which may involve Ca(2+) and play an important role in conferring cell sensitivity to apoptosis. Apoptosis may depend on both the presence of external apoptosis-activating signals, and, as shown in this study, on an internal factor represented by the ER.