D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function.

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function

A Novel Filarial Parasite (B. malayi) Stress-Activated Protein Kinase as a Potential Drug Target

Lymphatic filariasis (or elephantiasis) is a major neglected disease with an estimated 120 million individuals infected and approximately 1.5 billion at risk in endemic regions. It is a highly disfiguring and debilitating disease and one of the major causes of global morbidity. Treatment options for this disease are few and new drug targets and therapies need to be identified. We have identified a protein kinase ortholog of human p38 mitogen-activated protein kinase (p38) expressed in the filarial parasite, Brugia malayi (B. malayi), one of three causative agent of lymphatic filariasis. We hypothesize that this protein kinase, BmMPKl, is important for the organism’s growth and viability and as such, may be a novel therapeutic target. Human p38 plays an essential role in responses to stress, such as toxins, infection and inflammation as well as cell cycle control and apoptosis. An ortholog found in the nematode, Caenorhabditis elegans (C. elegans, PMK-1/2), plays a similar role in protecting the organism from oxidative stress. Elimination of PMK-1/2 through genetic means results in the inability of C. elegans to respond to oxidative stress, disrupts neuronal development, and innate immune responses. Based on these and other observations we hypothesize that BmMPKl plays a similar role in protecting B. malayi from oxidative stress and in parasite growth and development. The goals of this thesis project were: to produce recombinant BmMPKl kinase, assess the effects of known inhibitors of human p38 against BmMPKl, assess the effects of p38 inhibitors on B. malayi growth, replication, and response to oxidative stress

A rare case of posterior spinal cord syndrome following commando surgery: A case report and review of literature

A 60-year-old male who underwent commando surgery for oral cancer in a supine position and 20-degree neck extension developed sensory ataxia with a loss of proprioception in bilateral lower limbs and hands in the immediate postoperative period. The magnetic resonance imaging (MRI) of brain and screening of spine done within 6 h of surgery indicated a degenerative cervical canal stenosis from C3 to C7 level. A final diagnosis of posterior spinal cord syndrome (PCS) was made after excluding other causes clinically and radiologically. Emergency surgical decompression in the form of C3–C7 laminectomy and intravenous methylprednisolone were administered within 12 h of index surgery. An early diagnosis and treatment resulted in a good neurological recovery by the seventh postoperative day and he was ambulatory with minimal support at 3-month follow-up

D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function

The Role of a Brugia Malayi P38 MAP Kinase Ortholog (Bm-MPK1) in Parasite Anti-Oxidative Stress Responses

Filariasis, caused by thread-like nematode worms, affects millions of individuals throughout the tropics and is a major cause of acute and chronic morbidity. Filarial nematodes effectively evade host immunological responses and are long lived within their hosts. Recently an emphasis has been placed on enzymatic and non-enzymatic anti-oxidant systems which counteract the generation of reactive oxygen species (ROS) by macrophages and granulocytes, a first line of defense against parasites. We have characterized an anti-oxidant pathway in the filarial parasite Brugia malayi related to the evolutionarily conserved human mitogen-activated p38 protein kinase and the Caenorhabditis elegans PMK-1 protein kinase stress pathways. We have expressed a recombinant p38/PMK-1 ortholog from B. malayi (Bm-MPK1) and have successfully activated the kinase with mammalian upstream kinases. In addition, we have demonstrated inhibition of Bm-MPK1 activity using a panel of known p38 inhibitors. Using the potent and highly selective allosteric p38 inhibitor, BIRB796, we have implicated Bm-MPK1 in a pathway which offers B. malayi protection from the effects of ROS. Our results, for the first time, describe a stress-activated protein kinase pathway within the filarial parasite B. malayi which plays a role in protecting the parasite from ROS. Inhibition of this pathway may have therapeutic benefit in treating filariasis by increasing the sensitivity of filarial parasites to ROS and other reactive intermediates

Human Gut Bacteria Are Sensitive to Melatonin and Express Endogenous Circadian Rhythmicity

<div><p>Circadian rhythms are fundamental properties of most eukaryotes, but evidence of biological clocks that drive these rhythms in prokaryotes has been restricted to Cyanobacteria. In vertebrates, the gastrointestinal system expresses circadian patterns of gene expression, motility and secretion <i>in vivo</i> and <i>in vitro</i>, and recent studies suggest that the enteric microbiome is regulated by the host’s circadian clock. However, it is not clear how the host’s clock regulates the microbiome. Here, we demonstrate at least one species of commensal bacterium from the human gastrointestinal system, <i>Enterobacter aerogenes</i>, is sensitive to the neurohormone melatonin, which is secreted into the gastrointestinal lumen, and expresses circadian patterns of swarming and motility. Melatonin specifically increases the magnitude of swarming in cultures of <i>E</i>. <i>aerogenes</i>, but not in <i>Escherichia coli</i> or <i>Klebsiella pneumoniae</i>. The swarming appears to occur daily, and transformation of <i>E</i>. <i>aerogenes</i> with a flagellar motor-protein driven lux plasmid confirms a temperature-compensated circadian rhythm of luciferase activity, which is synchronized in the presence of melatonin. Altogether, these data demonstrate a circadian clock in a non-cyanobacterial prokaryote and suggest the human circadian system may regulate its microbiome through the entrainment of bacterial clocks.</p></div

Neither lab nor clinical strains of <i>E</i>. <i>coli</i> nor <i>K</i>. <i>pneumoniae</i> show swarming response to other indoles.

<p>Cultures of clinical isolates of <i>E</i>. <i>coli</i> (left panels, DH5-α (middle panels, and <i>K</i>. <i>pneumoniae</i> (right panels were tested for swarming/growth in the presence of tryptophan (top row, serotonin (middle row, and N-acetylserotonin (bottom row, n = 16 cultures per strain per treatment.</p

The data presented here show that a clinical isolate of <i>E</i>. <i>aerogenes</i> expresses a circadian rhythm in MotA expression and displays a swarming response to melatonin in a dose- and temperature-dependent manner.

<p>The data presented here show that a clinical isolate of <i>E</i>. <i>aerogenes</i> expresses a circadian rhythm in MotA expression and displays a swarming response to melatonin in a dose- and temperature-dependent manner.</p

Bioluminescence recording of <i>MotA</i>::<i>luxcdabe</i> transformed <i>E</i>. <i>aerogenes</i> confirms a temperature compensated circadian rhythm.

<p>A) Normalized bioluminescence rhythms from control-treated (top panels) and melatonin-treated (bottom panels) cultures show circadian rhythms at (from left to right) 27° (n = 5/treatment), 34° (n = 5/treatment), 37° (n = 5 control and 7 melatonin-treated) and 40° (n = 6 control and 6 melatonin-treated). Time scales represent days after plates were inoculated with bacteria, which varied in the amount of time needed to stabilize and begin outgrowth. B) Periodogram analysis-derived peak phases of rhythmic cultures from (A) reveal that control-treated cultures (white circles) show greater variation in peak phase than melatonin-treated cultures (black circles), which are more synchronized at all three temperatures. C) Periods of rhythms varied between 22 and 28 hours among temperature and melatonin treatments, but were not significantly affected by temperature or melatonin.</p