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1.  Rapid-thrombelastography (r-TEG) thresholds for goal-directed resuscitation of patients at risk for massive transfusion 
Uncontrolled hemorrhage is a leading cause of mortality following trauma accounting for up to 40% of deaths. Massive transfusion protocols (MTPs) offer a proven benefit in resuscitation of these patients. Recently, the superiority of thrombelastography (TEG)-guided resuscitation over strategies guided by conventional clotting assays (CCA) has been established. We seek to determine optimal thresholds for r-TEG driven resuscitation.
R-TEG data were reviewed for 190 patients presenting to our Level 1 Trauma Center from 2010 to 2015. Criteria for inclusion were highest level trauma activation in patients ≥ 18 years of age with hypotension presumed due to acute blood loss. Exclusion criteria included: isolated gun-shot wound to the head, pregnancy and chronic liver disease. Receiver operating characteristic (ROC) analysis was performed to test the predictive performance of r-TEG for massive transfusion requirement defined by need for 1) >10 units of RBCs total or death in the first six hours or 2) >4 units of RBCs in any hour within the first 6 hours. Cut-point analysis was then performed to determine optimal thresholds for TEG-based resuscitation.
ROC analysis of r-TEG yielded areas under the curve (AUC) greater than 70% for all outputs with respect to both transfusion thresholds considered, with exception of activated clotting time (ACT) and lysis at 30 minutes (LY30) for > 4U RBC in any hour in the first 6 hours. Optimal cut-point analysis of the resultant ROC curves was performed and for each value, the most sensitive cut-point was identified, respectively ACT ≥ 128 sec, angle (α) ≤ 65, maximum amplitude (MA) ≤ 55 mm and LY30 ≥ 5%.
Through ROC analysis of prospective TEG data, we have identified optimal thresholds to guide hemostatic resuscitation. These thresholds should be validated in a prospective multicenter trial.
Level of Evidence
Prognostic, level III
PMCID: PMC5177474  PMID: 27805995
Injury; Shock; Coagulopathy; Resuscitation; Transfusion
2.  The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase 
Structure (London, England : 1993)  2016;24(10):1658-1667.
Arginine kinase provides a model for functional dynamics, studied through crystallography, enzymology and NMR. Structures are now solved, at ambient temperature, for the transition state analog (TSA) complex. Analysis of quasi-rigid sub-domain displacements show that differences between the two TSA structures average about 5% of changes between substrate-free and TSA forms, with which they are nearly co-linear. Small backbone hinge rotations map to sites that also flex on substrate-binding. Anisotropic atomic displacement parameters (ADPs) are refined using rigid-body TLS constraints. Consistency between crystal forms shows that they reflect intrinsic molecular properties more than crystal lattice effects. In many regions, the favored directions of thermal/static displacement are appreciably correlated with movements on substrate-binding. Correlation between ADPs and larger substrate-associated movements implies that the latter approximately follow paths of low energy intrinsic motions.
Godsey et al. show that differences between two ambient temperature crystal forms of arginine kinase, and the orientations of TLS-constrained anisotropic B-factors, are directionally correlated to much larger displacements upon substrate-binding, indicating that binding-associated transitions take advantage of intrinsic modes of flexibility.
PMCID: PMC5052116  PMID: 27594681
ADP; Ambient; Anisotropic; Change; Conformational; Crystal; Dynamics; Induced-fit; Protein; Selection; Temperature
3.  Experience with High Dose Once-daily Vancomycin for Patients with Skin and Soft-tissue infections in an Ambulatory Setting 
Open Forum Infectious Diseases  2017;4(Suppl 1):S338.
Intravenous (IV) vancomycin is commonly used to treat MRSA (methicillin-resistant Staphylococcus aureus) infections and is typically dosed at 15 mg/kg every 12 hours. Pharmacokinetic studies, animal models, and limited human study suggest that once-daily high dose (30 mg/kg) vancomycin is similar in efficacy for certain types of infection in the hospital setting, but there is little evidence for its use in outpatient antibiotic therapy (OPAT).
We have used a high dose vancomycin regimen for MRSA skin and soft-tissue infections (SSTI) since 2011. We describe our experience with this regimen in an ambulatory setting supervised by Infectious Diseases (ID) physicians and evaluate its efficacy and safety.
This retrospective observational study included patients treated with vancomycin for SSTI from Jan 1, 2014 to July 31, 2015. Exclusion criteria included noncompliance with treatment. Patients with initial renal impairment were also excluded as they would be given 15 mg/kg vancomycin daily. Patient demographics, response to vancomycin, duration of therapy, readmission to emergency department (ER), and side effects experienced by patients were collected. A successful outcome was defined as no further requirement for IV vancomycin on the last day of therapy with either oral step down therapy or no further antibiotic.
407 charts were reviewed and 208 patients qualified for inclusion. The mean age of included patients was 38 years. Of the 208 patients, 31% were people who inject drugs, 39% had preceding SSTI in the 12 months prior, and 48% had been on antibiotics in the previous 8 weeks. Incision and drainage was done in 50% of the patients. There were 135 positive wound cultures, of which 58% grew MRSA. The average duration of treatment was 4.7 days.
A successful outcome was achieved in 162 patients (79%). Side effects including red man syndrome and phlebitis were seen in 19 patients (9.3%). 42 patients (20.5%) were readmitted to ER within 30 days of initial referral, and 18 of those were related to the original infections.
High dose (30 mg/kg) once-daily vancomycin for SSTI was effective and safe when used for select patients under the supervision of ID physicians in an ambulatory setting.
All authors: No reported disclosures.
PMCID: PMC5631202
4.  Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor 
Journal of Virology  2017;91(18):e00391-17.
Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.
IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.
PMCID: PMC5571256  PMID: 28679762
AAVR; adeno-associated virus; gene therapy; receptor-ligand interaction; viral receptor; virus overlay assay; virus-host interactions
5.  Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11 
Journal of Virology  2017;91(3):e01980-16.
Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.
IMPORTANCE Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) showed that assembly takes place in the nucleolus and is dependent on AAP and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to -12), we find that AAP is not an essential requirement for capsid assembly of AAV4, -5, and -11, and AAP, assembled capsids, and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotype-promiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly and show the heterogeneous nature of the assembly process within the AAV family.
PMCID: PMC5244341  PMID: 27852862
assembly-activating protein; adeno-associated virus; capsid assembly; gene therapy; nucleolus; parvovirus
6.  World Congress Integrative Medicine & Health 2017: Part one 
Brinkhaus, Benno | Falkenberg, Torkel | Haramati, Aviad | Willich, Stefan N. | Briggs, Josephine P. | Willcox, Merlin | Linde, Klaus | Theorell, Töres | Wong, Lisa M. | Dusek, Jeffrey | Wu, Darong | Eisenberg, David | Haramati, Aviad | Berger, Bettina | Kemper, Kathi | Stock-Schröer, Beate | Sützl-Klein, Hedda | Ferreri, Rosaria | Kaplan, Gary | Matthes, Harald | Rotter, Gabriele | Schiff, Elad | Arnon, Zahi | Hahn, Eckhard | Luberto, Christina M. | Martin, David | Schwarz, Silke | Tauschel, Diethard | Flower, Andrew | Gramminger, Harsha | Gupta, Hedwig H. | Gupta, S. N. | Kerckhoff, Annette | Kessler, Christian S. | Michalsen, Andreas | Kessler, Christian S. | Kim, Eun S. | Jang, Eun H. | Kim, Rana | Jan, Sae B. | Mittwede, Martin | Mohme, Wiebke | Ben-Arye, Eran | Bonucci, Massimo | Saad, Bashar | Breitkreuz, Thomas | Rossi, Elio | Kebudi, Rejin | Daher, Michel | Razaq, Samaher | Gafer, Nahla | Nimri, Omar | Hablas, Mohamed | Kienle, Gunver Sophia | Samuels, Noah | Silbermann, Michael | Bandelin, Lena | Lang, Anna-Lena | Wartner, Eva | Holtermann, Christoph | Binstock, Maxwell | Riebau, Robert | Mujkanovic, Edin | Cramer, Holger | Lauche, Romy | Michalsen, Andres | Ward, Lesley | Cramer, Holger | Irnich, Dominik | Stör, Wolfram | Burnstock, Geoffrey | Schaible, Hans-Georg | Ots, Thomas | Langhorst, Jost | Lauche, Romy | Sundberg, Tobias | Falkenberg, Torkel | Amarell, Catherina | Amarell, Catherina | Anheyer, Melanie | Eckert, Marion | Eckert, Marion | Ogal, Mercedes | Eckert, Marion | Amarell, Catherina | Schönauer, Annette | Reisenberger, Birgit | Brand, Bernhard | Anheyer, Dennis | Dobos, Gustav | Kroez, Matthias | Martin, David | Matthes, Harald | Ammendola, Aldo | Mao, Jun J. | Witt, Claudia | Yang, Yufei | Dobos, Gustav | Oritz, Miriam | Horneber, Markus | Voiß, Petra | Reisenberger, Birgit | von Rosenstiel, Alexandra | Eckert, Marion | Ogal, Mercedes | Amarell, Catharina | Anheyer, Melanie | Schad, Friedemann | Schläppi, Marc | Kröz, Matthias | Büssing, Arndt | Bar-Sela, Gil | Matthes, Harald | Schiff, Elad | Ben-Arye, Eran | Arnon, Zahi | Avshalomov, David | Attias, Samuel | Schönauer, Annette | Haramati, Aviad | Witt, Claudia | Brinkhaus, Benno | Cotton, Sian | Jong, Miek | Jong, Mats | Scheffer, Christian | Haramati, Aviad | Tauschel, Diethard | Edelhäuser, Friedrich | AlBedah, Abdullah | Lee, Myeong Soo | Khalil, Mohamed | Ogawa, Keiko | Motoo, Yoshiharu | Arimitsu, Junsuke | Ogawa, Masao | Shimizu, Genki | Stange, Rainer | Kraft, Karin | Kuchta, Kenny | Watanabe, Kenji | Bonin, D | Büssing, Arndt | Gruber, Harald | Koch, Sabine | Gruber, Harald | Pohlmann, Urs | Caldwell, Christine | Krantz, Barbara | Kortum, Ria | Martin, Lily | Wieland, Lisa S. | Kligler, Ben | Gould-Fogerite, Susan | Zhang, Yuqing | Wieland, Lisa S. | Riva, John J. | Lumpkin, Michael | Ratner, Emily | Ping, Liu | Jian, Pei | Hamme, Gesa-Meyer | Mao, Xiaosong | Chouping, Han | Schröder, Sven | Hummelsberger, Josef | Wullinger, Michael | Brodzky, Marc | Zalpour, Christoff | Langley, Julia | Weber, Wendy | Mudd, Lanay M. | Wayne, Peter | Witt, Clauda | Weidenhammer, Wolfgang | Fønnebø, Vinjar | Boon, Heather | Steel, Amie | Bugarcic, Andrea | Rangitakatu, Melisa | Steel, Amie | Adams, Jon | Sibbritt, David | Wardle, Jon | Leach, Matthew | Schloss, Janet | Dieze, Helene | Boon, Heather | Ijaz, Nadine | Willcox, Merlin | Heinrich, Michael | Lewith, George | Flower, Andrew | Graz, Bertrand | Adam, Daniela | Grabenhenrich, Linus | Ortiz, Miriam | Binting, Sylvia | Reinhold, Thomas | Brinkhaus, Benno | Andermo, Susanne | Sundberg, Tobias | Falkenberg, Torkel | Nordberg, Johanna Hök | Arman, Maria | Bhasin, Manoj | Fan, Xueyi | Libermann, Towia | Fricchione, Gregory | Denninger, John | Benson, Herbert | Berger, Bettina | Stange, Rainer | Michalsen, Andreas | Martin, David D. | Boers, Inge | Vlieger, Arine | Jong, Miek | Brinkhaus, Benno | Teut, Michael | Ullmann, Alexander | Ortiz, Miriam | Rotter, Gabriele | Binting, Sylvia | Lotz, Fabian | Roll, Stephanie | Canella, Claudia | Mikolasek, Michael | Rostock, Matthias | Beyer, Jörg | Guckenberger, Matthias | Jenewein, Josef | Linka, Esther | Six, Claudia | Stoll, Sarah | Stupp, Roger | Witt, Claudia M. | Chuang, Elisabeth | Kligler, Ben | McKee, Melissa D. | Cramer, Holger | Lauche, Romy | Klose, Petra | Lange, Silke | Langhorst, Jost | Dobos, Gustav | Chung, Vincent C. H. | Wong, Hoi L. C. | Wu, Xin Y. | Wen, Grace Y. G. | Ho, Robin S. T. | Ching, Jessica Y. L. | Wu, Justin C. Y. | Coakley, Amanda | Flanagan, Jane | Annese, Christine | Empoliti, Joanne | Gao, Zishan | Liu, Xugang | Yu, Shuguang | Yan, Xianzhong | Liang, Fanrong | Hohmann, Christoph D. | Steckhan, Nico | Ostermann, Thomas | Paetow, Arion | Hoff, Evelyn | Michalsen, Andreas | Hu, Xiao-Yang | Wu, Ruo-Han | Logue, Martin | Blonde, Clara | Lai, Lily Y. | Stuart, Beth | Flower, Andrew | Fei, Yu-Tong | Moore, Michael | Liu, Jian-Ping | Lewith, George | Hu, Xiao-Yang | Wu, Ruo-Han | Logue, Martin | Blonde, Clara | Lai, Lily Y. | Stuart, Beth | Flower, Andrew | Fei, Yu-Tong | Moore, Michael | Liu, Jian-Ping | Lewith, George | Jeitler, Michael | Zillgen, Hannah | Högl, Manuel | Steckhan, Nico | Stöckigt, Barbara | Seifert, Georg | Michalsen, Andreas | Kessler, Christian | Khadivzadeh, Talat | Bashtian, Maryam Hassanzadeh | Aval, Shapour Badiee | Esmaily, Habibollah | Kim, Jihye | Kim, Keun H. | Klocke, Carina | Joos, Stefanie | Koshak, Abdulrahman | Wie, Li | Koshak, Emad | Wali, Siraj | Alamoudi, Omer | Demerdash, Abdulrahman | Qutub, Majdy | Pushparaj, Peter | Heinrich, Michael | Kruse, Sigrid | Fischer, Isabell | Tremel, Nadine | Rosenecker, Joseph | Leung, Brenda | Takeda, Wendy | Liang, Ning | Feng, Xue | Liu, Jian-ping | Cao, Hui-juan | Luberto, Christina M. | Shinday, Nina | Philpotts, Lisa | Park, Elyse | Fricchione, Gregory L. | Yeh, Gloria | Munk, Niki | Zakeresfahani, Arash | Foote, Trevor R. | Ralston, Rick | Boulanger, Karen | Özbe, Dominik | Gräßel, Elmar | Luttenberger, Katharina | Pendergrass, Anna | Pach, Daniel | Bellmann-Strobl, Judit | Chang, Yinhui | Pasura, Laura | Liu, Bin | Jäger, Sven F. | Loerch, Ronny | Jin, Li | Brinkhaus, Benno | Ortiz, Miriam | Reinhold, Thomas | Roll, Stephanie | Binting, Sylvia | Icke, Katja | Shi, Xuemin | Paul, Friedemann | Witt, Claudia M. | Rütz, Michaela | Lynen, Andreas | Schömitz, Meike | Vahle, Maik | Salomon, Nir | Lang, Alon | Lahat, Adi | Kopylov, Uri | Ben-Horin, Shomron | Har-Noi, Ofir | Avidan, Benjamin | Elyakim, Rami | Gamus, Dorit | NG, Siew | Chang, Jessica | Wu, Justin | Kaimiklotis, John | Schumann, Dania | Buttó, Ludovica | Langhorst, Jost | Dobos, Gustav | Haller, Dirk | Cramer, Holger | Smith, Caroline | de Lacey, Sheryl | Chapman, Michael | Ratcliffe, Julie | Johnson, Neil | Lyttleton, Jane | Boothroyd, Clare | Fahey, Paul | Tjaden, Bram | van Vliet, Marja | van Wietmarschen, Herman | Jong, Miek | Tröger, Wilfried | Vuolanto, Pia | Aarva, Paulina | Sorsa, Minna | Helin, Kaija | Wenzel, Claudia | Zoderer, Iris | Pammer, Patricia | Simon, Patrick | Tucek, Gerhard | Wode, Kathrin | Henriksson, Roger | Sharp, Lena | Stoltenberg, Anna | Nordberg, Johanna Hök | Xiao-ying, Yang | Wang, Li-qiong | Li, Jin-gen | Liang, Ning | Wang, Ying | Liu, Jian-ping | Balneaves, Lynda | Capler, Rielle | Bocci, Chiara | Guffi, Marta | Paolini, Marina | Meaglia, Ilaria | Porcu, Patrizia | Ivaldi, Giovanni B. | Dragan, Simona | Bucuras, Petru | Pah, Ana M. | Badalica-Petrescu, Marius | Buleu, Florina | Hogea-Stoichescu, Gheorghe | Christodorescu, Ruxandra | Kao, Lan | Cho, Yumin | Klafke, Nadja | Mahler, Cornelia | von Hagens, Cornelia | Uhlmann, Lorenz | Bentner, Martina | Schneeweiss, Andreas | Mueller, Andreas | Szecsenyi, Joachim | Joos, Stefanie | Neri, Isabella | Ortiz, Miriam | Schnabel, Katharina | Teut, Michael | Rotter, Gabriele | Binting, Sylvia | Cree, Margit | Lotz, Fabian | Suhr, Ralf | Brinkhaus, Benno | Rossi, Elio | Baccetti, Sonia | Firenzuoli, Fabio | Monechi, Maria V. | Di Stefano, Mariella | Amunni, Gianni | Wong, Wendy | Chen, Bingzhong | Wu, Justin | Amri, Hakima | Haramati, Aviad | Kotlyanskaya, Lucy | Anderson, Belinda | Evans, Roni | Kligler, Ben | Marantz, Paul | Bradley, Ryan | Booth-LaForce, Cathryn | Zwickey, Heather | Kligler, Benjamin | Brooks, Audrey | Kreitzer, Mary J. | Lebensohn, Patricia | Goldblatt, Elisabeth | Esmel-Esmel, Neus | Jiménez-Herrera, Maria | Ijaz, Nadine | Boon, Heather | Jocham, Alexandra | Stock-Schröer, Beate | Berberat, Pascal O. | Schneider, Antonius | Linde, Klaus | Masetti, Morgana | Murakozy, Henriette | Van Vliet, Marja | Jong, Mats | Jong, Miek | Agdal, Rita | Atarzadeh, Fatemeh | Jaladat, Amir M. | Hoseini, Leila | Amini, Fatemeh | Bai, Chen | Liu, Tiegang | Zheng, Zian | Wan, Yuxiang | Xu, Jingnan | Wang, Xuan | Yu, He | Gu, Xiaohong | Daneshfard, Babak | Nimrouzi, Majid | Tafazoli, Vahid | Alorizi, Seyed M. Emami | Saghebi, Seyed A. | Fattahi, Mohammad R. | Salehi, Alireza | Rezaeizadeh, Hossein | Zarshenas, Mohammad M. | Nimrouzi, Majid | Fox, Kealoha | Hughes, John | Kostanjsek, Nenad | Espinosa, Stéphane | Lewith, George | Fisher, Peter | Latif, Abdul | Lefeber, Donald | Paske, William | Öztürk, Ali Ö. | Öztürk, Gizemnur | Boers, Inge | Tissing, Wim | Naafs, Marianne | Busch, Martine | Jong, Miek | Daneshfard, Babak | Sanaye, Mohammad R. | Dräger, Kilian | Fisher, Peter | Kreitzer, Mary J. | Evans, Roni | Leininger, Brent | Shafto, Kate | Breen, Jenny | Sanaye, Mohammad R. | Daneshfard, Babak | Simões-Wüst, Ana P. | Moltó-Puigmartí, Carolina | van Dongen, Martien | Dagnelie, Pieter | Thijs, Carel | White, Shelley | Wiesener, Solveig | Salamonsen, Anita | Stub, Trine | Fønnebø, Vinjar | Abanades, Sergio | Blanco, Mar | Masllorens, Laia | Sala, Roser | Al-Ahnoumy, Shafekah | Han, Dongwoon | He, Luzhu | Kim, Ha Yun | In Choi, Da | Alræk, Terje | Stub, Trine | Kristoffersen, Agnete | von Sceidt, Christel | Michalsen, Andreas | Bruset, Stig | Musial, Frauke | Anheyer, Dennis | Cramer, Holger | Lauche, Romy | Saha, Felix J. | Dobos, Gustav | Anheyer, Dennis | Haller, Heidemarie | Lauche, Romy | Dobos, Gustav | Cramer, Holger | Azizi, Hoda | Khadem, Nayereh | Hassanzadeh, Malihe | Estiri, Nazanin | Azizi, Hamideh | Tavassoli, Fatemeh | Lotfalizadeh, Marzieh | Zabihi, Reza | Esmaily, Habibollah | Azizi, Hoda | Shabestari, Mahmoud Mohammadzadeh | Paeizi, Reza | Azari, Masoumeh Alvandi | Bahrami-Taghanaki, Hamidreza | Zabihi, Reza | Azizi, Hamideh | Esmaily, Habibollah | Baars, Erik | De Bruin, Anja | Ponstein, Anne | Baccetti, Sonia | Di Stefano, Mariella | Rossi, Elio | Firenzuoli, Fabio | Segantini, Sergio | Monechi, Maria Valeria | Voller, Fabio | Barth, Jürgen | Kern, Alexandra | Lüthi, Sebastian | Witt, Claudia | Barth, Jürgen | Zieger, Anja | Otto, Fabius | Witt, Claudia | Beccia, Ariel | Dunlap, Corina | Courneene, Brendan | Bedregal, Paula | Passi, Alvaro | Rodríguez, Alfredo | Chang, Mayling | Gutiérrez, Soledad | Beissner, Florian | Beissner, Florian | Preibisch, Christine | Schweizer-Arau, Annemarie | Popovici, Roxana | Meissner, Karin | Beljanski, Sylvie | Belland, Laura | Rivera-Reyes, Laura | Hwang, Ula | Berger, Bettina | Sethe, Dominik | Hilgard, Dörte | Heusser, Peter | Bishop, Felicity | Al-Abbadey, Miznah | Bradbury, Katherine | Carnes, Dawn | Dimitrov, Borislav | Fawkes, Carol | Foster, Jo | MacPherson, Hugh | Roberts, Lisa | Yardley, Lucy | Lewith, George | Bishop, Felicity | Al-Abbadey, Miznah | Bradbury, Katherine | Carnes, Dawn | Dimitrov, Borislav | Fawkes, Carol | Foster, Jo | MacPherson, Hugh | Roberts, Lisa | Yardley, Lucy | Lewith, George | Bishop, Felicity | Holmes, Michelle | Lewith, George | Yardley, Lucy | Little, Paul | Cooper, Cyrus | Bogani, Patrizia | Maggini, Valentina | Gallo, Eugenia | Miceli, Elisangela | Biffi, Sauro | Mengoni, Alessio | Fani, Renato | Firenzuoli, Fabio | Brands-Guendling, Nadine | Guendling, Peter W. | Bronfort, Gert | Evans, Roni | Haas, Mitch | Leininger, Brent | Schulz, Craig | Bu, Xiangwei | Wang, J. | Fang, T. | Shen, Z. | He, Y. | Zhang, X. | Zhang, Zhengju | Wang, Dali | Meng, Fengxian | Büssing, Arndt | Baumann, Klaus | Frick, Eckhard | Jacobs, Christoph | Büssing, Arndt | Grünther, Ralph-Achim | Lötzke, Désirée | Büssing, Arndt | Jung, Sonny | Lötzke, Désirée | Recchia, Daniela R. | Robens, Sibylle | Ostermann, Thomas | Berger, Bettina | Stankewitz, Josephin | Kröz, Matthias | Jeitler, Mika | Kessler, Christian | Michalsen, Andreas | Cheon, Chunhoo | Jang, Bo H. | Ko, Seong G. | Huang, Ching W. | Sasaki, Yui | Ko, Youme | Cheshire, Anna | Ridge, Damien | Hughes, John | Peters, David | Panagioti, Maria | Simon, Chantal | Lewith, George | Cho, Hyun J. | Han, Dongwoon | Choi, Soo J. | Jung, Young S. | Im, Hyea B | Cooley, Kieran | Tummon-Simmons, Laura | Cotton, Sian | Luberto, Christina M. | Wasson, Rachel | Kraemer, Kristen | Sears, Richard | Hueber, Carly | Derk, Gwendolyn | Lill, JR | An, Ruopeng | Steinberg, Lois | Rodriguez, Lourdes Diaz | la Fuente, Francisca García-de | De la Vega, Miguel | Vargas-Román, Keyla | Fernández-Ruiz, Jonatan | Cantarero-Villanueva, Irene | Rodriguez, Lourdes Diaz | García-De la Fuente, Francisca | Jiménez-Guerrero, Fanny | Vargas-Román, Keyla | Fernández-Ruiz, Jonatan | Galiano-Castillo, Noelia | Diaz-Saez, Gualberto | Torres-Jimenez, José I. | Garcia-Gomez, Olga | Hortal-Muñoz, Luis | Diaz-Diez, Camino | Dicen, Demijon | Diezel, Helene | Adams, Jon | Steel, Amie | Wardle, Jon | Diezel, Helene | Steel, Amie | Frawley, Jane | Wardle, Jon | Broom, Alex | Adams, Jon | Dong, Fei | Yu, He | Liu, Tiegang | Ma, Xueyan | Yan, Liyi | Wan, Yuxiang | Zheng, Zian | Gu, Xiaohong | Dong, Fei | Yu, He | Wu, Liqun | Liu, Tiegang | Ma, Xueyan | Ma, Jiaju | Yan, Liyi | Wan, Yuxiang | Zheng, Zian | Zhen, Jianhua | Gu, Xiaohong | Dubois, Julie | Rodondi, Pierre-Yves | Edelhäuser, Friedrich | Schwartze, Sophia | Trapp, Barbara | Cysarz, Dirk
PMCID: PMC5498855
8.  Goal-directed Hemostatic Resuscitation of Trauma-induced Coagulopathy 
Annals of surgery  2016;263(6):1051-1059.
Massive transfusion protocols (MTPs) have become standard of care in the management of bleeding injured patients, yet strategies to guide them vary widely. We conducted a pragmatic, randomized clinical trial (RCT) to test the hypothesis that an MTP goal directed by the viscoelastic assay thrombelastography (TEG) improves survival compared with an MTP guided by conventional coagulation assays (CCA).
This RCT enrolled injured patients from an academic level-1 trauma center meeting criteria for MTP activation. Upon MTP activation, patients were randomized to be managed either by an MTP goal directed by TEG or by CCA (ie, international normalized ratio, fibrinogen, platelet count). Primary outcome was 28-day survival.
One hundred eleven patients were included in an intent-to-treat analysis (TEG = 56, CCA = 55). Survival in the TEG group was significantly higher than the CCA group (log-rank P = 0.032, Wilcoxon P = 0.027); 20 deaths in the CCA group (36.4%) compared with 11 in the TEG group (19.6%) (P = 0.049). Most deaths occurred within the first 6 hours from arrival (21.8% CCA group vs 7.1% TEG group) (P = 0.032). CCA patients required similar number of red blood cell units as the TEG patients [CCA: 5.0 (2–11), TEG: 4.5 (2–8)] (P = 0.317), but more plasma units [CCA: 2.0 (0–4), TEG: 0.0 (0–3)] (P = 0.022), and more platelets units [CCA: 0.0 (0–1), TEG: 0.0 (0–0)] (P = 0.041) in the first 2 hours of resuscitation.
Utilization of a goal-directed, TEG-guided MTP to resuscitate severely injured patients improves survival compared with an MTP guided by CCA and utilizes less plasma and platelet transfusions during the early phase of resuscitation.
PMCID: PMC5432433  PMID: 26720428
coagulopathy; fibrinolysis; goal-directed; resuscitation; thrombelastography; transfusion
9.  The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide 
Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ’s directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.
Graphical Abstract
PMCID: PMC5415311
AAV; adeno-associated virus; glycan; heparan sulfate; attachment; receptor; structure; electron microscopy; gene therapy; vector
10.  Retrospective evidence on outcomes and experiences of pregnancy and childbirth in epidermolysis bullosa in Australia and New Zealand☆☆☆★ 
Pregnancy in epidermolysis bullosa (EB) has not been comprehensively studied.
We aimed to develop a foundational database, which could provide peri-obstetric advice in EB.
Survey questionnaires were sent to obstetricians, unaffected mothers of EB babies, and mothers with EB. Results were analyzed using chi-square, Fisher exact, and t-tests.
Out of 1346 obstetricians surveyed, 195 responded, and only 14 had encountered EB. All recommended normal vaginal delivery (NVD), except for one elective Caesarean section (CS). We received responses from 75 unaffected mothers who had delivered EB babies. They had significantly more complications in their EB pregnancies compared to their non-EB pregnancies. A further 44 women with various types of EB who had given birth responded. Most delivered via NVD and had no significant increase in complications in both their EB and non-EB pregnancies. In both groups, there were no significant differences in blistering at birth in babies delivered via NVD and CS.
In conclusion, most patients with EB who are capable of giving birth do not have an increased risk for pregnancy-related complications and NVD appears to be safe. Awareness of this data amongst obstetricians and dermatologists should lead to improved quality of care for mothers and babies affected with EB.
Capsule Summary
•There is limited information on pregnancy in EB.•This is the first comprehensive study in the world on pregnancy experiences of a large group of mothers with EB, unaffected mothers of EB babies and an obstetrician survey.•We have recommendations for mothers expecting EB babies and expectant mothers with EB.
PMCID: PMC5418959
Epidermolysis Bullosa; Pregnancy; Delivery; Childbirth; Anesthesia
11.  High hospital research participation and improved colorectal cancer survival outcomes: a population-based study 
Gut  2016;66(1):89-96.
In 2001, the National Institute for Health Research Cancer Research Network (NCRN) was established, leading to a rapid increase in clinical research activity across the English NHS. Using colorectal cancer (CRC) as an example, we test the hypothesis that high, sustained hospital-level participation in interventional clinical trials improves outcomes for all patients with CRC managed in those research-intensive hospitals.
Data for patients diagnosed with CRC in England in 2001–2008 (n=209 968) were linked with data on accrual to NCRN CRC studies (n=30 998). Hospital Trusts were categorised by the proportion of patients accrued to interventional studies annually. Multivariable models investigated the relationship between 30-day postoperative mortality and 5-year survival and the level and duration of study participation.
Most of the Trusts achieving high participation were district general hospitals and the effects were not limited to cancer ‘centres of excellence’, although such centres do make substantial contributions. Patients treated in Trusts with high research participation (≥16%) in their year of diagnosis had lower postoperative mortality (p<0.001) and improved survival (p<0.001) after adjustment for casemix and hospital-level variables. The effects increased with sustained research participation, with a reduction in postoperative mortality of 1.5% (6.5%–5%, p<2.2×10−6) and an improvement in survival (p<10−19; 5-year difference: 3.8% (41.0%–44.8%)) comparing high participation for ≥4 years with 0 years.
There is a strong independent association between survival and participation in interventional clinical studies for all patients with CRC treated in the hospital study participants. Improvement precedes and increases with the level and years of sustained participation.
PMCID: PMC5256392  PMID: 27797935
12.  Overwhelming tPA Release, not PAI-1 Degradation, is Responsible for Hyperfibrinolysis in Severely Injured Trauma Patients 
Trauma induced coagulopathy (TIC) is associated with a four-fold increased risk of mortality. Hyperfibrinolysis is a component of TIC, but its mechanism is poorly understood. PAI-1 degradation by activated protein C has been proposed as mechanism for deregulation of the plasmin system in hemorrhagic shock, but in other settings of ischemia, tPA has been shown to be elevated. We hypothesized that the hyperfibrinolysis in TIC is not the result of PAI-1 degradation, but is driven by an increase in tPA, with resultant loss of PAI-1 activity through complexation with tPA.
86 consecutive trauma activation patients had blood collected at the earliest time after injury, and were screened for hyperfibrinolysis using thrombelastography (TEG). Twenty-five hyperfibrinolytic patients were compared to 14 healthy controls using ELISAs for active tPA, active PAI-1 and PAI-1/tPA complex. Blood was also subjected to TEG with exogenous tPA-challenge as a functional assay for PAI-1 reserve.
Total levels of PAI-1 (the sum of the active PAI-1 species and its covalent complex with tPA) are not significantly different between hyperfibrinolytic trauma patients and healthy controls: median 104 pM (IQR 48—201 pM) versus 115 pM (IQR 54—202 pM). The ratio of active to complexed PAI-1, however, was two orders of magnitude lower in hyperfibrinolysis than controls. Conversely, total tPA levels (active plus complex) were significantly higher in hyperfibrinolysis than controls: 139 pM (IQR 68—237 pM) versus 32 pM (IQR 16—37 pM). Hyperfibrinolytic trauma patients displayed increased sensitivity to exogenous challenge with tPA: median LY30 of 66.8% compared to 9.6% for controls.
Depletion of PAI-1 in TIC is driven by an increase in tPA, not PAI-1 degradation. The tPA-challenged TEG, based on this principle, is a functional test for PAI-1 reserves. Exploration of the mechanism of upregulation of tPA is critical to an understanding of hyperfibrinolysis in trauma.
PMCID: PMC4688194  PMID: 26491796
hyperfibrinolysis; PAI-1; tPA; aPC; trauma; massive transfusion
13.  Diagnosis of pancreaticobiliary malignancy by detection of minichromosome maintenance protein 5 in biliary brush cytology 
British Journal of Cancer  2017;116(3):349-355.
Biliary brush cytology is the standard method of evaluating biliary strictures, but is insensitive at detecting malignancy. In pancreaticobiliary cancer minichromosome maintenance replication proteins (MCM 2–7) are dysregulated in the biliary epithelium and MCM5 levels are elevated in bile samples. This study aimed to validate an immunocolorimetric ELISA assay for MCM5 as a pancreaticobiliary cancer biomarker in biliary brush samples.
Biliary brush specimens were collected prospectively at ERCP from patients with a biliary stricture. Collected samples were frozen at −80 °C. The supernatant was washed and lysed cells incubated with HRP-labelled anti-MCM5 mouse monoclonal antibody. Test positivity was determined by optical density absorbance. Patients underwent biliary brush cytology or additional investigations as per clinical routine.
Ninety-seven patients were included in the study; 50 had malignant strictures. Median age was 65 years (range 21–94) and 51 were male. Compared with final diagnosis the MCM5 assay had a sensitivity for malignancy of 65.4% compared with 25.0% for cytology. In the 72 patients with paired MCM5 assay and biliary brush cytology, MCM5 demonstrated an improved sensitivity (55.6% vs 25.0% P=0.0002) for the detection of malignancy.
Minichromosome maintenance replication protein5 is a more sensitive indicator of pancreaticobiliary malignancy than standard biliary brush cytology.
PMCID: PMC5294492  PMID: 28081547
pancreatic ductal adenocarcinoma; cholangiocarcinoma; endoscopic retrograde cholangiopancreaticography; chronic pancreatitis; primary sclerosing cholangitis; IGG4-related disease; minichromosome maintenance replication proteins
14.  The “Black Diamond”: Rapid TEG Identifies Lethal Hyperfibrinolysis 
Post-injury hyperfibrinolysis (HF), defined as LY30 ≥3% on rapid thrombelastography (rTEG), is associated with high mortality and large utilization of blood products. We observed that some cases of HF are reversible and are associated with patients who respond to hemostatic resuscitation; whereas, other cases of severe HF appear to be associated with these patients’ inevitable demise. We therefore sought to define this unsurvivable subtype of hyperfibrinolysis as a recognizable rTEG tracing pattern.
We queried our trauma registry for patients who either died or spent at least one day in the ICU, received at least one unit of PRBCs, and had an admission rTEG. Within this group of 572 patients, we identified 42 pairs of non-survivors and survivors who matched on age, sex, injury mechanism and NISS. We inspected the rTEG tracings to ascertain if any pattern was found exclusively within the non-surviving group and applied these findings to the cohort of 572 patients to assess the predictive value for mortality.
Within the matched group 17% of patients developed HF. Within the HF subgroup, a unique rTEG pattern was present in 14 HF patients who died and in none of the survivors. This pattern was a “diamond-shaped” tracing with a short time to maximum amplitude (TMA) of ≤14 min and complete lysis before the LY30 point. Applying these criteria to the 572 unmatched patients, this pattern had a 100% positive predictive value for mortality.
Patients displaying the “black diamond” pattern on their admission rTEG are at higher risk for mortality. Given the volume of blood products and other resources that these patients consume, this TEG pattern may represent an objective criterion to discontinue efforts at hemostatic resuscitation.
PMCID: PMC4684985  PMID: 26488324
hyperfibrinolysis; mortality; thrombelastography; trauma induced coagulopathy; hemorrhagic shock
15.  Shock-Induced Systemic Hyperfibrinolysis is Attenuated by Plasma First Resuscitation 
We developed a hemorrhagic shock animal model to replicate an urban prehospital setting where resuscitation fluids are limited to assess the effect of saline versus plasma in coagulopathic patients. An in vitro model of whole blood dilution with saline exacerbated tPA mediated fibrinolysis, while plasma dilution did not change fibrinolysis. We hypothesize that shock induced hyperfibrinolysis can be attenuated by resuscitation with plasma while exacerbated by saline.
Sprague Dawley rats were hemorrhaged to a mean arterial pressure (MAP) of 25 mmHg and maintained in shock for 30 minutes. Animals were resuscitated with either normal saline (NS) or platelet free plasma (PFP) with a 10% total blood volume bolus, followed by an additional 5 minutes of resuscitation with NS to increase blood pressure to a MAP of 30 mmHg. Animals were observed for 15 minutes for assessment of hemodynamic response and survival. Blood samples were analyzed with thrombelastography (TEG) paired with protein analysis.
The median percent of total blood volume shed per group were similar (NS 52.5% vs PFP 55.7 p=0.065). Survival was 50% in NS compared to 100% in PFP. The change in LY30 and tPA levels from baseline to shock was similar between groups (LY30 PFP10 IQR 4.3 to 11.2, NS 4.5 IQR 4.1 to 14.2 p=1.00; tPA PFP 16.6 ng/ml IQR 13.7–27.8, NS 22.4 IQR 20.1 to 25.5 p=0.240). After resuscitation, the median change in LY30 was greater in the NS group (13.5 IQR 3.5 to 19.9) compared to PFP (−4.9 % IQR −9.22 to 0.25 p=0.004) but tPA levels did not significantly change (NS 1.4 IQR-6.2 to 7.1 vs PFP 1.7 IQR-5.2 to 6.8 p=0.699).
Systemic hyperfibrinolysis is driven by hypoperfusion and associated with increased levels of tPA. Plasma is a superior resuscitation fluid to normal saline in a prehospital model of severe hemorrhagic shock as it attenuates hyperfibrinolysis and improves systemic perfusion.
PMCID: PMC4686159  PMID: 26680132
Hyperfibrinolysis; Plasma first resuscitation; Hemorrhagic shock
16.  The Influence of Frame Alignment with Dose Compensation on the Quality of Single Particle Reconstructions 
Journal of structural biology  2015;192(2):196-203.
As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR coupled with their high-throughput frame collection abilities. In approaching atomic resolution of native-like biomolecules, the accurate extraction of structural locations and orientations of side-chains from frames depends not only on the electron dose that a sample receives but also on the ability to accurately estimate the CTF. Here we use a new 2.8 Å resolution structure of a recombinant gene therapy virus, AAV-DJ with Arixtra, imaged on an FEI Titan Krios with a DE-20 Direct Electron detector to probe new metrics including relative side-chain density and ResLog analysis for optimizing the compensation of electron beam damage and to characterize the factors that are limiting the resolution of the reconstruction. The influence of dose compensation on the accuracy of CTF estimation and particle classifiability are also presented. We show that rigorous dose compensation allows for better particle classifiability and greater recovery of structural information from negatively charged, electron-sensitive side-chains, resulting in a more accurate macromolecular model.
PMCID: PMC4633374  PMID: 26391007
Direct electron detector; Radiation damage; Beam-induced motion; Dose compensation; Atomic resolution; Cryo-electron microscopy
17.  A Straightforward Electrochemical Approach to Imine‐ and Amine‐bisphenolate Metal Complexes with Facile Control Over Metal Oxidation State 
ChemistryOpen  2016;5(4):351-356.
Synthetic methods to prepare organometallic and coordination compounds such as Schiff‐base complexes are diverse, with the route chosen being dependent upon many factors such as metal–ligand combination and metal oxidation state. In this work we have shown that electrochemical methodology can be employed to synthesize a variety of metal–salen/salan complexes which comprise diverse metal–ligand combinations and oxidation states. Broad application has been demonstrated through the preparation of 34 complexes under mild and ambient conditions. Unprecedented control over metal oxidation state (MII/III/IV where M=Fe, Mn) is presented by simple modification of reaction conditions. Along this route, a general protocol‐switch is described which allows access to analytically pure FeII/III–salen complexes. Tuning electrochemical potential, selective metalation of a Mn/Ni alloy is also presented which exclusively delivers MnII/IV–salen complexes in high yield.
PMCID: PMC4981056  PMID: 27547645
electrochemistry; organometallic compounds; salen; Schiff bases; synthesis
18.  Fibrinolysis Shutdown Phenotype Masks Changes in Rodent Coagulation in Tissue Injury versus Hemorrhagic Shock 
Surgery  2015;158(2):386-392.
Systemic hyperfibrinolysis (accelerated clot degradation) and fibrinolysis shutdown (impaired clot degradation) are associated with increased mortality compared to physiologic fibrinolysis following trauma. Animal models have not reproduced these changes. We hypothesize rodents have a shutdown phenotype that require an exogenous profibrinolytic to differentiate mechanisms that promote or inhibit fibrinolysis.
Fibrinolysis resistance was assessed by thrombelastography (TEG) using exogenous tissue plasminogen activator (tPA) titrations in whole blood. There were three experimental groups: 1)tissue injury (laparotomy/bowel crush), 2)shock (hemorrhage to mean arterial pressure 20 mmHg), and 3)control (arterial cannulation and tracheostomy). Baseline and 30-minute post-intervention blood samples were collected, and assayed with TEG challenged with taurocholic acid (TUCA).
Rats were resistant to exogenous tPA; CL30 (Percent clot remaining 30 minutes after maximum amplitude) at 150ng/ml (p=0.511) and 300ng/ml (p=0.931) was similar to baseline, while 600ng/ml (p=0.046) provoked fibrinolysis. Using the TUCA challenge, the percent change in CL30 from baseline was increased in tissue injury compared to control (p=0.048.), whereas CL30 decreased in shock versus control (p=0.048). TPA increased in the shock group compared to tissue injury (p=0.009) and control (p=0.012).
Rats have an innate fibrinolysis shutdown phenotype. The TEG TUCA challenge is capable of differentiating changes in clot stability with rats undergoing different procedures. Tissue injury inhibits fibrinolysis while shock promotes tPA-mediated fibrinolysis.
PMCID: PMC4492895  PMID: 25979440
19.  COMBAT: Initial experience with a randomized clinical trial of plasma-based resuscitation in the field for traumatic hemorrhagic shock 
Shock (Augusta, Ga.)  2015;44(0 1):63-70.
The existing evidence shows great promise for plasma as the first resuscitation fluid in both civilian and military trauma. We embarked on the Control of Major Bleeding After Trauma (COMBAT) trial with the support of the Department of Defense, in order to determine if plasma-first resuscitation yields hemostatic and survival benefits. The methodology of the COMBAT study represents not only three years of development work, but the integration of nearly two-decades of technical experience with the design and implementation of other clinical trials and studies. Herein, we describe the key features of the study design, critical personnel and infrastructural elements, and key innovations. We will also briefly outline the systems engineering challenges entailed by this study. COMBAT is a randomized, placebo controlled, semi-blinded prospective Phase IIB clinical trial, conducted in a ground ambulance fleet based at a Level I trauma center, and part of a multicenter collaboration. The primary objective of COMBAT is to determine the efficacy of field resuscitation with plasma first, compared to standard of care (normal saline). To date we have enrolled 30 subjects in the COMBAT study. The ability to achieve intervention with a hemostatic resuscitation agent in the closest possible temporal proximity to injury is critical and represents an opportunity to forestall the evolution of the “bloody vicious cycle”. Thus, the COMBAT model for deploying plasma in first response units should serve as a model for RCTs of other hemostatic resuscitative agents.
PMCID: PMC4504796  PMID: 25784527
trauma induced coagulopathy; plasma first resuscitation; hemostatic resuscitation; COMBAT; randomized clinical trial; trauma; hemorrhage; hemorrhagic shock; plasma; FFP; FP24
20.  A Straightforward Electrochemical Approach to Imine‐ and Amine‐bisphenolate Metal Complexes with Facile Control Over Metal Oxidation State 
ChemistryOpen  2016;5(4):351-356.
Synthetic methods to prepare organometallic and coordination compounds such as Schiff‐base complexes are diverse, with the route chosen being dependent upon many factors such as metal–ligand combination and metal oxidation state. In this work we have shown that electrochemical methodology can be employed to synthesize a variety of metal–salen/salan complexes which comprise diverse metal–ligand combinations and oxidation states. Broad application has been demonstrated through the preparation of 34 complexes under mild and ambient conditions. Unprecedented control over metal oxidation state (MII/III/IV where M=Fe, Mn) is presented by simple modification of reaction conditions. Along this route, a general protocol‐switch is described which allows access to analytically pure FeII/III–salen complexes. Tuning electrochemical potential, selective metalation of a Mn/Ni alloy is also presented which exclusively delivers MnII/IV–salen complexes in high yield.
PMCID: PMC4981056  PMID: 27547645
electrochemistry; organometallic compounds; salen; Schiff bases; synthesis
21.  Parsimony in Protein Conformational Change 
Structure (London, England : 1993)  2015;23(7):1190-1198.
Protein conformational change is analyzed by finding the minimalist backbone torsion angle rotations that superpose crystal structures within experimental error. Of several approaches to enforcing parsimony during flexible least-squares superposition, an ℓ1-norm restraint provided greatest consistency with independent indications of flexibility from NMR relaxation dispersion and chemical shift perturbation in arginine kinase, and four previously studied systems. Crystallographic cross-validation shows that the dihedral parameterization describes conformational change more accurately than rigid-group approaches. The rotations that superpose the principal elements of structure constitute a small fraction of the raw (φ, ψ)-differences that also reflect local conformation and experimental error. Substantial long-range displacements can be mediated by modest dihedral rotations, accommodated even within α-helices and β-sheets without disruption of hydrogen bonding at the hinges. Consistency between ligand-associated and intrinsic motions (in the unliganded state) implies that induced changes tend to follow low-barrier paths between conformational sub-states that are in intrinsic dynamic equilibrium.
PMCID: PMC4497923  PMID: 26095029
22.  Postinjury Fibrinolysis Shutdown: Rationale for Selective Tranexamic Acid 
The journal of trauma and acute care surgery  2015;78(6 0 1 0 0 0 0 0 6):S65-S69.
Postinjury systemic fibrinolysis has been recognized as a biologic process for more than 200 years, but the specific mechanisms of regulation and their clinical implications remain to be elucidated. By the 1950s, the plasminogen-plasmin-antiplasmin system was established as critical in preserving microvascular patency during blood clotting to maintain hemostasis. The challenges in modulating systemic fibrinolysis became evident soon thereafter. In the 1960s systemic fibrinolysis was identified by thrombelastography (TEG) during the anhepatic phase of liver transplantation, prompting the recommendation for intraoperative antifibrinolytics. But the administration of antifibrinolytic was associated with fatal postoperative pulmonary emboli. During the same period, there was experimental evidence that antifibrinolytics prevented irreversible hemorrhagic shock. More recently, a randomized trial indicated that plasmin inhibition during coronary artery bypass grafting was associated with increased mortality. The interest in antifibrinolytic therapy for trauma induced coagulopathy (TIC) is a relatively recent event, largely driven by the increasing use of viscoelastic hemostatic assays. The CRASH-2 trial, published in 2010, stimulated worldwide enthusiasm for tranexamic acid (TXA). However, the limitations of this study were soon acknowledged, raising concern for the unbridled use of TXA. Most recently, the documentation of fibrinolysis shutdown soon after injury has highlighted the potential adverse effects due to the untimely administration of TXA. A recent retrospective analysis in severely injured patients supports this hypothesis. But final clarity of this volatile topic awaits the completion of the current ongoing randomized clinical trials throughout the world.
PMCID: PMC4443855  PMID: 26002266
23.  Plasma Is the Physiologic Buffer of tPA Mediated Fibrinolysis: Rationale for Plasma First Resuscitation after Life-Threatening Hemorrhage 
Pre-hospital resuscitation with crystalloid exacerbates fibrinolysis, which is associated with high mortality. We hypothesize that plasma compared to crystalloid resuscitation prevents hyperfibrinolysis in a tissue plasminogen activator (tPA) rich environment via preservation of proteins essential for regulation of fibrinolysis.
Study Design
Healthy individuals donated blood, which was assayed using a native (non activated) thrombelastography (TEG). Whole-blood (WB) was mixed with normal saline (NS) or platelet poor plasma (PPP) at progressive dilutions. TPA was added to promote a fibrinolytic environment. In a separate experiment PPP was run through 100 KD filter and liquid remaining on top of the filter (TFP) and below the filter (BFP) was obtained. Whole blood was diluted by 50% with TFP, BPF and NS and assayed with tPA TEG challenge. TFP and BFP were assayed for protein concentration and protein composition.
NS and PPP dilution of WB with out tPA did not affect clot lysis at 30 minutes (LY30) (NS Spearman’s Rho 0.300 p=0.186 and PPP 0.294 p=0.288). When tPA was added NS dilution of whole blood increased LY30 in a percentage dependent manner (0.844 p<0.001) but did not significantly increase with PPP dilution (0.270 p=0.202). The difference in LY30 from WB to diluted WB with PPP (mean change −1.05 95% CI −9.42 to 7.33) was similar with TFP (1.23 95%CI −5.20 to 7.66 p=0.992). However, both BPF (37.65 95%CI 24.47 to 50.82 p=0.001) and NS (47.36 95%CI 34.3–60.45 p<0.001) showed large increases in fibrinolysis compared to PPP.
Crystalloid and plasma dilution of whole blood does not increase fibrinolysis. However NS dilution of WB, increases susceptibility to tPA mediated fibrinolysis. Plasma resuscitation, simulated by plasma dilution of whole blood, attenuates increased susceptibility to tPA mediate fibrinolysis. The benefits of plasma resuscitation are mediated through preservation of plasma proteins.
PMCID: PMC4409505  PMID: 25840538
24.  Thrombelastographic Pattern Recognition in Renal Disease and Trauma 
Thrombelastography (TEG) is a viscoelastic hemostatic assay. We have observed that end stage renal disease (ESRD) and trauma induced coagulopathy (TIC) produce distinctive TEG tracings. We hypothesized that rigorously definable TEG patterns could discriminate between healthy controls and patients with ESRD and TIC.
TEG was performed on blood from ESRD patients (n=54) and blood from trauma patients requiring a massive blood transfusion (n=16). Plots of independent TEG parameters were analyzed for patterns coupled to disease state, compared to controls. Decision trees for taxonomic classification were then built using the “R-Project” statistical software.
Minimally overlapping clusters of TEG results were observed for the three patient groups when coordinate pairs of maximum amplitude (MA) and TEG activated clotting time (ACT) were plotted on orthogonal axes. Based upon these groupings, a taxonomical classification tree was constructed using MA and TEG ACT. Branch points were set at an ACT of 103 seconds, and these branches subdivided for MA at 60.8 mm for the high ACT branch and 72.6 mm for the low ACT branch, providing a correct classification rate of 93.4%.
ESRD and TIC demonstrate distinct TEG patterns. The coagulopathy of ESRD is typified by a prolonged enzymatic phase of clot formation, with normal-to-elevated final clot strength. Conversely, TIC is typified by prolonged clot formation and weakened clot strength. Our taxonomic categorization constitutes a rigorous system for the algorithmic interpretation of TEG based upon cluster analysis. This will form the basis for clinical decision support software for viscoelastic hemostatic assays.
PMCID: PMC4346387  PMID: 25577141
machine learning; pattern recognition; renal disease; trauma; coagulopathy; hypercoagulability; trauma induced coagulopathy; thrombelastography; viscoelastic hemostatic assay; classification tree analysis; taxonomy; hemodialysis
25.  Laparoscopy-assisted ERCP (LA-ERCP) following bariatric gastric bypass surgery: initial experience of a single UK centre 
Frontline Gastroenterology  2015;7(1):54-59.
Bariatric gastric bypass surgery is being increasingly performed, but endoscopic retrograde cholangiopancreatography (ERCP) in these patients poses a unique challenge because of a lack of per-oral access to the stomach. Small series suggest a higher technical success rate using laparoscopy-assisted ERCP (LA-ERCP) than with an enteroscopic approach via the Roux-en-Y anastomosis. We present initial experience of LA-ERCP in our unit.
Retrospective case series of consecutive patients undergoing LA-ERCP in our unit between September 2011 and July 2014. Data was retrieved from electronic, clinical and endoscopy records.
Seven LA-ERCPs were performed. All seven patients were female, with median age 44 years (range 36–71). Indications included symptomatic bile duct stones (5/7), benign papillary fibrosis (1/7) and retained biliary stent (1/7). 5/7 (71%) patients had had a prior cholecystectomy. To facilitate LA-ERCP, laparoscopic gastrostomy ports were created in all patients. Duodenal access, biliary cannulation and completion of therapeutic aim were achieved in all patients. 6/7 (86%) patients required endoscopic sphincterotomy. The median duration of procedures was 94 min (range 70–135). Median postoperative length of stay was 2 days (range 1–9). One patient developed mild postprocedural acute pancreatitis, and another patient developed a mild port-site infection. Otherwise, no procedure-related complications were seen. All patients remained well on follow-up (median 14 months (range 1–35) from date of ERCP), with no evidence of further biliary symptoms.
Our early experience of LA-ERCP is that it is safe and effective. The technique may require particular consideration, as bariatric surgery is increasingly performed, in a patient group at significant risk of bile duct stones.
PMCID: PMC5369458

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