The crystal structure has been determined of the F 1 -catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F 1 -ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the e-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the e-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α 3 β 3 -catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the e-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F 1 -ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated e-subunit unable to bind ATP, the enzyme remains inactive and the e-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the e-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α 3 β 3 -catalytic domain is likewise closely similar to those of active F 1 -ATPases. However, although the β E -catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

Gulf War Illness (GWI) is a chronic multisymptom condition with a central nervous system (CNS) component, for which there is no treatment available. It is now believed that the combined exposure to Gulf War (GW) agents, including pyridostigmine bromide (PB) and pesticides, such as permethrin (PER), was a key contributor to the etiology of GWI. In this study, a proteomic approach was used to characterize the biomolecular disturbances that accompany neurobehavioral and neuropathological changes associated with combined exposure to PB and PER. Mice acutely exposed to PB and PER over 10 days showed an increase in anxiety-like behavior, psychomotor problems and delayed cognitive impairment compared to control mice that received vehicle only. Proteomic analysis showed changes in proteins associated with lipid metabolism and molecular transport in the brains of GW agent-exposed mice compared to controls. Proteins associated with the endocrine and immune systems were also altered, and dysfunction of these systems is a prominent feature of GWI. The presence of astrogliosis in the GW agent-exposed mice compared to control mice further suggests an immune system imbalance, as is observed in GWI. These studies provide a broad perspective of the molecular disturbances driving the late pathology of this complex illness. Evaluation of the potential role of these biological functions in GWI will be useful in identifying molecular pathways that can be targeted for the development of novel therapeutics against GWI.

The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, “On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.”

ObjectiveExposure to repetitive concussion, or mild traumatic brain injury (mTBI), has been linked with increased risk of long‐term neurodegenerative changes, specifically chronic traumatic encephalopathy (CTE). To date, preclinical studies largely have focused on the immediate aftermath of mTBI, with no literature on the lifelong consequences of mTBI in these models. This study provides the first account of lifelong neurobehavioral and histological consequences of repetitive mTBI providing unique insight into the constellation of evolving and ongoing pathologies with late survival.MethodsMale C57BL/6J mice (aged 2–3 months) were exposed to either single or repetitive mild TBI or sham procedure. Thereafter, animals were monitored and assessed at 24 months post last injury for measures of motor coordination, learning deficits, cognitive function, and anxiety‐like behavior prior to euthanasia and preparation of the brains for detailed neuropathological and protein biochemical studies.ResultsAt 24 months survival animals exposed to r‐mTBI showed clear evidence of learning and working memory impairment with a lack of spatial memory and vestibule‐motor vestibulomotor deficits compared to sham animals. Associated with these late behavioral deficits there was evidence of ongoing axonal degeneration and neuroinflammation in subcortical white matter tracts. Notably, these changes were also observed after a single mTBI, albeit to a lesser degree than repetitive mTBI.InterpretationIn this context, our current data demonstrate, for the first time, that rather than an acute, time limited event, mild TBI can precipitate a lifelong degenerative process. These data therefore suggest that successful treatment strategies should consider both the acute and chronic nature of mTBI.

Phospholipid (PL) abnormalities are observed in the cerebrospinal fluid of patients with traumatic brain injury (TBI), suggesting their role in TBI pathology. Therefore, PL levels were examined in a TBI mouse model that received 1.8 mm deep controlled cortical impact injury or craniectomy only (control). The rotarod and Barnes maze acquisition and probe tests were performed within 2 wk after injury, with another probe test performed 3 mo postinjury. Liquid chromatography/mass spectrometry analyses were performed on lipid extracts from several brain regions and plasma from injured and control mice collected at 3 mo postinjury. Compared to controls, injured mice with sensorimotor and learning deficits had decreased levels of cortical and cerebellar phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels, while hippocampal PC, sphingomyelin and PE levels were elevated. Ether PE levels were lower in the cortices and plasma of injured animals. Polyunsaturated fatty acid-containing PC and PE species, particularly ratios of docosahexaenoic acid (DHA) to arachidonic acid, were lower in the hippocampi and cortices and plasma of injured mice. Given the importance of DHA in maintaining neuronal function and resolving inflammation and of peroxisomes in synthesis of ether PLs, normalizing these PLs may be a useful strategy for treating the chronic pathology of TBI.-Abdullah, L., Evans, J. E., Ferguson, S., Mouzon, B., Montague, H., Reed, J., Crynen, G., Emmerich, T., Crocker, M., Pelot, R., Mullan, M., Crawford, F. Lipidomic analyses identify injury-specific phospholipid changes 3 months after traumatic brain injury. FASEB J. 28, 5311-5321 (2014). www.fasebj.org Key Words: controlled cortical impact ⅐ mass spectrometry ⅐ phosphatidylcholine ⅐ phosphatidylethanolamine ⅐ phosphatidylinositol ⅐ sphingomyelin Traumatic brain injury (TBI) is a significant cause of morbidity and mortality in the United States among adults from both the civilian and military populations. Due to the improvements made in military body armor and combat helmets over the past 2 decades, the rate of severe TBI associated with Iraq and Afghanistan deployments has decreased to 1% of the total TBI sustained by military personnel, a rate that is lower than those reported for previous deployments (1-3). Estimates from the U.S. Centers for Disease Control and Prevention suggest that severe TBI remains an important issue in the civilian population. In 2010, 2.5 million admissions to emergency departments were due to TBI (4) contributing to about a third of injury related deaths in the United States. The annual U.S. costs associated with TBI are estimated to be $75 billion, largely attributed to medical care, loss of productivity, and long-term disability (5). Although mild TBI occurs more frequently than severe TBI, the latter has the largest adverse effect on the activities of daily living of injured individuals and accounts for 90% of the total TBI-related medical costs (5). The primary injury is caused by the immediate force of the trauma a...

The Na ؉ -translocating F-ATPase of the thermoalkaliphilic bacterium Clostridium paradoxum harbors an oligomeric ring of c subunits that resists dissociation by sodium dodecyl sulfate. The c ring has been isolated and crystallized in two dimensions. From electron microscopy of these c-ring crystals, a projection map was calculated to 7 Å resolution. In the projection map, each c ring consists of two concentric, slightly staggered, packed rings, each composed of 11 densities representing the ␣-helices. On the basis of these results, it was determined that the F-ATPase from C. paradoxum contains an undecameric c ring.The greatest challenge to alkaliphilic bacteria living at extreme pH values (i.e., ϾpH 9) is the maintenance of their intracellular pH near neutral in order to prevent cytoplasmic alkalinization. These bacteria maintain a ⌬pH of 1.5 to 2.5 units (acid inside), which opposes the bioenergetics of ATP synthesis using a H ϩ -coupled F 1 F o -ATP synthase. Due to the inverted pH gradient, the total proton motive force (⌬H ϩ ) may drop to values well below Ϫ100 mV, which is not sufficient for ATP synthesis by conventional mechanisms. Clostridium paradoxum is an anaerobic thermoalkaliphile that ferments glucose rapidly to acetate, ethanol, and CO 2 and produces ATP by substrate phosphorylation (3, 10). C. paradoxum has recently been reported to contain a Na ϩ -translocating F 1 F oATPase, and several of the enzyme's properties suggest that its primary operation is that of an ATP-driven Na ϩ ion pump rather than that of an ATP synthase (7). The net result of the pumping is the formation of an electrochemical gradient of sodium ions (⌬Na ϩ ), which is purportedly used to drive bioenergetic processes (e.g., transport and motility).The F o subcomplex of F 1 F o -ATP synthases is involved in trafficking ions across the membrane. Either the electrochemical ion gradient across the membrane produces torque and drives ATP synthesis or ATP hydrolysis drives the active transport of the ions. An assembly of c subunits in the membrane forms an hourglass-shaped rotary cylinder, known as the c ring. As has been documented for the Na ϩ -dependent ATP synthases of other bacteria (1, 9, 15), the ATP synthase of C. paradoxum contains an oligomeric c ring that is stable on sodium dodecyl sulfate (SDS)-polyacrylamide gels (7) and is therefore well-suited for structural analysis (12). Structural data on c-ring stoichiometries have been determined only for Na ϩ -ATP synthases that operate under physiological conditions (e.g., during succinate or tartrate fermentation) in the ATP synthesis direction (6) and for a Na ϩ -translocating VATPase that operates in the ATP hydrolysis direction (13). In this communication, we present the structural analysis of the c ring isolated from the ATP-hydrolyzing Na ϩ -ATPase of C. paradoxum and show that the F o rotor is assembled as an undecameric c ring. MATERIALS AND METHODSBacterial strain and culture conditions. Clostridium paradoxum DSM 7308 was maintained as a spore stock preparation in ster...

In the military population, there is high comorbidity between mild traumatic brain injury (mTBI) and post-traumatic stress disorder (PTSD) due to the inherent risk of psychological trauma associated with combat. These disorders present with long-term neurological dysfunction and remain difficult to diagnose due to their comorbidity and overlapping clinical presentation. Therefore, we performed cross-sectional analysis of blood samples from demographically matched soldiers (total, n = 120) with mTBI, PTSD, and mTBI+PTSD and those who were considered cognitively and psychologically normal. Soldiers were genotyped for apolipoprotein E (APOE) ɛ4, and phospholipids (PL) were examined using liquid chromatography/mass spectrometry analysis. We observed significantly lower levels of several major PL classes in TBI, PTSD, and TBI+PTSD, compared with controls. PTSD severity analysis revealed that significant PL decreases were primarily restricted to the moderate-to-severe PTSD group. An examination of the degree of unsaturation showed that monounsaturated fatty acid-containing phosphatidylcholine (PC) and phosphatidylinositol (PI) species were lower in the TBI and TBI+PTSD groups. However, these PLs were unaltered among PTSD subjects, compared with controls. Similarly, ether PC (ePC) levels were lower in PTSD and TBI+PTSD subjects, relative to controls. Ratios of arachidonic acid (AA) to docosahexaenoic acid (DHA)-containing species were significantly decreased within PC and phosphatidylethanolamine (PE) classes. APOE ɛ4 (+) subjects exhibited higher PL levels than their APOE ɛ4 (-) counterparts within the same diagnostic groups. These findings suggest that PL profiles, together with APOE genotyping, could potentially aid to differentiate diagnosis of mTBI and PTSD and warrant further validation. In conclusion, PL profiling may facilitate clinical diagnosis of mTBI and PTSD currently hindered by comorbid pathology and overlapping symptomology of these two conditions.

Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56°C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum. The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an F 1 F o -ATP synthase but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q 28 , E 61 , and S 62 ). On the basis of these properties, the F 1 F o -ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of Na ؉ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the subunit that has been shown to be essential for coupled ATP synthesis.