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11.
Bacteriophages (hence termed phages) are viruses that target bacteria and have long been considered as potential future treatments against antibiotic-resistant bacterial infection. However, the molecular nature of phage interactions with bacteria and the human host has remained elusive for decades, limiting their therapeutic application. While many phages and their functional repertoires remain unknown, the advent of next-generation sequencing has increasingly enabled researchers to decode new lytic and lysogenic mechanisms by which they attack and destroy bacteria. Furthermore, the last decade has witnessed a renewed interest in the utilization of phages as therapeutic vectors and as a means of targeting pathogenic or commensal bacteria or inducing immunomodulation. Importantly, the narrow host range, immense antibacterial repertoire, and ease of manipulating phages may potentially allow for their use as targeted modulators of pathogenic, commensal and pathobiont members of the microbiome, thereby impacting mammalian physiology and immunity along mucosal surfaces in health and in microbiome-associated diseases. In this review, we aim to highlight recent advances in phage biology and how a mechanistic understanding of phage–bacteria–host interactions may facilitate the development of novel phage-based therapeutics. We provide an overview of the challenges of the therapeutic use of phages and how these could be addressed for future use of phages as specific modulators of the human microbiome in a variety of infectious and noncommunicable human diseases. 相似文献
12.
A new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF) 总被引:2,自引:0,他引:2
A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high- resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands: a more intense central one and four less intense, two moving faster and two slower than the central band. In normal platelets, each multimer could also be resolved into five bands. The central one had a mobility similar to that in plasma, whereas the four satellite bands had a mobility that differed from that of the corresponding plasma bands. In the patient, platelet von Willebrand factor antigen content and ristocetin cofactor activity were normal, and von Willebrand factor showed the same structure of individual multimers as seen in normal platelets. On the other hand, plasma von Willebrand factor antigen and ristocetin cofactor activity were decreased, and the structure of individual von Willebrand factor multimers was different from that of normal plasma and similar to that seen in normal and patient's platelets. After infusion of 1-deamino-8-D-arginine vasopressin, the largest von Willebrand factor multimers, as well as new satellite bands with a mobility similar to those in normal plasma, appeared in the patient plasma, and the levels of von Willebrand factor antigen and ristocetin cofactor activity became normal. Yet no relevant change in the prolonged bleeding time was observed. This new variant of von Willebrand disease, therefore, is characterized by the presence of a dysfunctional von Willebrand factor molecule that exhibits unique structural abnormalities in plasma but appears to be normal in platelets. The designation of Type IIF is proposed for this type of von Willebrand disease in accordance with the terminology that has been previously used. 相似文献
13.
14.
Castaman G Lethagen S Federici AB Tosetto A Goodeve A Budde U Batlle J Meyer D Mazurier C Fressinaud E Goudemand J Eikenboom J Schneppenheim R Ingerslev J Vorlova Z Habart D Holmberg L Pasi J Hill F Peake I Rodeghiero F 《Blood》2008,111(7):3531-3539
We have prospectively evaluated the biologic response to desmopressin in 77 patients with type 1 von Willebrand disease (VWD) enrolled within the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD project. Complete response to desmopressin was defined as an increase of both ristocetin cofactor activity (VWF:RCo) and factor VIII coagulant activity (FVIII:C) to 50 IU/dL or higher and partial response as VWF:RCo or FVIII:C lower than 50 IU/dL after infusion, but at least 3-fold the basal level. Complete response was observed in 83% of patients; partial in 13%; and no response in 4%. Patients with some abnormality of VWF multimeric pattern had significantly lower basal FVIII:C and VWF, lower VWF:RCo/Ag ratio, and less complete responses to desmopressin than patients with a normal multimeric pattern (P=.002). Patients with mutations at codons 1130 and 1205 in the D'-D3 domain had the greatest relative increase, but shortest FVIII and VWF half-lives after infusion. Most partial and nonresponsive patients had mutations in the A1-A3 domains. Response to desmopressin in these VWD patients seemed to be associated with the location of the causative mutation. The presence of subtle multimeric abnormalities did not hamper potential clinically useful responses, as in typical type 1 VWD. 相似文献
15.
Federici AB 《Haematologica》2003,88(6):EREP02
Factor VIIII (FVIII) and von Willebrand factor (VWF) are two distinct but related glycoproteins that circulate in plasma as a tightly bound complex (FVIII/VWF). Their deficiencies or structural defects are responsible for the most common inherited bleeding disorders, namely hemophilia A (HA) and von Willebrand's disease (VWD). The VWF has a dual role in hemostasis: first it promotes platelet adhesion to thrombogenic surfaces as well as platelet-to-platelet cohesion during thrombus formation; second, it is the carrier for FVIII in plasma. FVIII acts as a co-factor to accelerate the activation of factor X by activated factor IX in the coagulation cascade. After many years of investigations, the molecular mechanisms of FVIII/VWF interactions are now well known and recent biochemical investigations have confirmed that VWF is a key partner for FVIII, playing significant roles in FVIII function, its production and its stabilization, in its conformation and immunogenicity. FVIII and VWF are both present in most plasma-derived FVIII/VWF concentrates used in clinical practice. FVIII/VWF concentrates can be classified into three main categories according to the degree of their purification. Intermediate-high purity plasma-derived concentrates containing FVIII/VWF currently in use since 1987 carry a low risk of transmitting blood-borne infections. Concentrate safety depends on the interaction of two factors: the decrease of viral plasma load and the increase of viral inactivation. These FVIII/VWF concentrates are currently used in type 3 VWD and in type 1 or 2 VWD patients who are unresponsive to desmopressin (DDAVP). More recently the presence of the physiologic FVIII/VWF complex has been considered to play an important role also in replacement therapy for patients with HA. The correct use of FVIII/VWF concentrates in VWD and HA have been reported in several national and international guidelines. 相似文献
16.
Giuseppe Stabile Antoine Lepillier Ermenegildo De Ruvo Marco Scaglione Matteo Anselmino Frederic Sebag Domenico Pecora Mark Gallagher Mariano Rillo Graziana Viola Luca Rossi Valerio De Santis Maurizio Landolina Antonello Castro Massimo Grimaldi Nicolas Badenco Maurizio Del Greco Antonio De Simone Ennio Pisan Salim Abbey Filippo Lamberti Antonio Pani Giulio Zucchelli Giuseppe Sgarito Daniela Dugo Emanuele Bertaglia Teresa Strisciuglio Francesco Solimene 《Journal of cardiovascular electrophysiology》2020,31(7):1694-1701
17.
Filippo Torroni Erminia Romeo Francesca Rea Paola De Angelis Francesca Foschia Simona Faraci Giovanni Federici di Abriola Anna Chiara Contini Tamara Caldaro Luigi Dall’Oglio 《World journal of gastrointestinal endoscopy》2014,6(7):318-323
AIM: To assess the usefulness of the balloon assisted enteroscopy in preventing surgical intervention in pa-tients with Peutz-Jeghers syndrome (PJS) having a small bowel large polyps. METHODS: Seven consecutive asymptomatic pts(age 15-38 years) with PJS have been collected; six under-went polypectomy using single balloon enteroscopy(Olympus SIF Q180) with antegrade approach using push and pull technique. SBE system consists of the SIF-Q180 enteroscope, an overtube balloon control unit(OBCU Olympus Balloon Control Unit) and a dispos-able silicone splinting tube with balloon(ST-SB1). All procedures were performed under general anesthesia. Previously all pts received wireless capsule endos-copy(WCE). Prophylactic polypectomy was reservedmainly in pts who had polyps 15 mm in diameter. The balloon is inflated and deflated by a balloon control unit with a safety pressure setting range from-6.0 kPa to +5.4 kPa. Informed consent has been obtained from pts or parents for each procedure.RESULTS: Six pts underwent polypectomy of small bowel polyps; in 5 pts a large polyp 15 mm(range 20-50 mm in diameter) was resected; in 1 patient with WCE negative, SBE was performed for previous surgi-cal resection of gastrointestinal stromal tumors. In 2 pts endoscopic clips were placed due to a polypectomy. No surgical complication have been reported. SBE with resection of small bowel large polyps in PJS pts was useful to avoid gastrointestinal bleeding and emergency laparotomy due to intestinal intussuscep-tions. No gastrointestinal tumors were found in sub-sequent enteroscopic surveillance in all seven pts. In order surveillance, all pts received WCE, upper en-doscopy, ileocolonoscopy every 2 years. No pts had extraintestinal malignant lesions. SBE was performed when WCE was positive for significant polyps( 15 mm).CONCLUSION: The effective of prophylactic polyp-ectomy of small bowel large polyps( 15 mm) could be the first line treatment for conservative approach in management of PJS patients. 相似文献
18.
Michael R. Sawaya Duilio Cascio Mari Gingery Jose Rodriguez Lukasz Goldschmidt Jacques-Philippe Colletier Marc M. Messerschmidt Sébastien Boutet Jason E. Koglin Garth J. Williams Aaron S. Brewster Karol Nass Johan Hattne Sabine Botha R. Bruce Doak Robert L. Shoeman Daniel P. DePonte Hyun-Woo Park Brian A. Federici Nicholas K. Sauter Ilme Schlichting David S. Eisenberg 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(35):12769-12774
It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.The advent of X-ray free-electron lasers (XFELs) has made it possible to obtain atomic resolution macromolecular structures from crystals with sizes approximating only 1/60th of the volume of a single red blood cell. Brief, intense pulses of coherent X-rays, focused on a spot of 3-μm diameter, have produced 1.9-Å-resolution diffraction data from a stream of lysozyme crystals, each crystal no bigger than 3 μm3 (1). A stream of crystals, not just one crystal, is required to collect the many tens of thousands of diffraction patterns that compose a complete data set. No single crystal can contribute more than one diffraction pattern because the XFEL beam is so intense and the crystals so small that the crystals are typically vaporized after a single pulse. Impressively, a photosystem I crystal no bigger than 10 unit cells (300 nm) on an edge produced observable subsidiary diffraction peaks between Bragg reflections, details which would be unobservable from conventionally sized crystals (2). With this new ability to collect diffraction patterns from crystals of unprecedentedly small dimensions, it is conceivable that high-resolution diffraction data could be collected from crystals in vivo. The structure obtained in this manner would be unaltered from that occurring naturally in a living cell, free from distortion that might otherwise potentially arise from nonphysiological conditions imposed by recrystallization. A practical advantage would also be gained by eliminating the need for a protein purification step, whether the in vivo grown crystals were naturally, or heterologously expressed (3).The nascent field of serial femtosecond crystallography (SFX) has published results on nine different macromolecular systems since its inception in 2009 (3, 9). The crystals for this study were not grown in artificial crystallization chambers as has been the protocol of conventional macromolecular crystallography since the 1950s. Instead, crystals were grown in cells. Specifically, they were grown in Sf9 insect cells, heterologously expressing Trypanosoma brucei cathepsin B. These in vivo-grown crystals were used for the XFEL diffraction experiment. To this end, the cells were lysed and the crystals were extracted before injecting them in the XFEL beam for data collection. This last purification step seems to be the only major departure from our goal of obtaining high-resolution structural information from crystal inclusions in vivo, without requiring the crystal to be extracted from the cell that assembled it. Here we attempt to go one step further than previous studies—to record diffraction from crystals within living cells.
Open in a separate window*The available XFEL energy was limited to 2 keV (6.2 Å wavelength) when these experiments were conducted.Our target for in vivo crystal structure determination is the insecticidal Cry3A toxin from Bacillus thuringiensis (Bt). The bacterium naturally produces crystals of toxin during sporulation (16). Presumably, the capacity for in vivo crystallization evolved in Bt as a mechanism to store the toxin in a concentrated, space-efficient manner. Since the 1920s, farmers have used the crystalline insecticidal proteins to control insect pests; its production as a natural pesticide is now a commercial enterprise. Attempts to structurally characterize the toxins date back to more than 40 y ago with the first report of diffraction from isolated crystals that were packed together in powder form to obtain a measurable signal; X-ray sources available at the time were relatively weak (17). More than 20 y later, the structure was determined at 2.5-Å resolution by single crystal diffraction using a synchrotron X-ray source (18). However, to achieve this result, the authors dissolved the naturally occurring microcrystals and recrystallized the toxin using the hanging drop vapor diffusion method. To date, more than a dozen Bt toxin structures have been reported from various strains [Protein Data Bank (PDB) ID codes 1cby, 1ciy, 1i5p, 1ji6, 1w99, 2d42, 2c9k, 2rci, 3eb7, 2ztb, 3ron, 4d8m, 4ato, 4ary, and 4arx], but none using naturally occurring crystals, and all of the crystals had lost their native context.In pursuit of in vivo diffraction, we took advantage of the Bt subsp. israelensis strain 4Q7/pPFT3As to produce the largest in vivo crystals achievable. This strain contains the plasmid pPFT3As, which increases expression of Cry3A by 12.7-fold over wild type by using strong promoters and an mRNA stabilizing sequence (19). The level of Cry3A production is such that the cell essentially distorts to take on the shape of the enclosed crystal. The calculated average crystal volume is 0.7 µm3 (19), almost accounting for the volume of the cell. To explore the possibilities for in situ data collection of in vivo microcrystals, we injected both the crystals in cells and crystals that we isolated from cells in the XFEL beam and collected SFX diffraction data. Our experiments revealed that the cell wall and other cellular components are not an obstacle to achieving 2.9-Å-resolution diffraction, and analogous studies in other systems might be similarly successful. 相似文献
Table 1.
SFX publications from XFEL sources to datePublication date | System | Product | Resolution (Å) | Title of publication | Authors | Reference |
Feb 2011* | Photosystem I | Structure | 8.7 | Femtosecond X-ray protein nanocrystallography | Chapman et al. | 2 |
Dec 2011* | Lysozyme | Structure | 8.7 | Radiation damage in protein serial femtosecond crystallography using an X-ray free-electron laser | Lomb et al. | 4 |
Jan 2012* | Photosystem I-Ferredoxin | Data | 11 | Time-resolved protein nanocrystallography using an X-ray free-electron laser | Aquila et al. | 5 |
Jan 2012* | Cathepsin B | Data | 7.5 | In vivo protein crystallization opens new routes in structural biology | Koopman et al. | 3 |
Jan 2012* | Photosynthetic Reaction Center | Structure | 7.4 | Lipidic phase membrane protein serial femtosecond crystallography | Johansson et al. | 6 |
Jun 2012 | Photosystem II | Structure | 6.6 | Room temperature femtosecond X-ray diffraction of photosystem II microcrystals | Kern et al. | 7 |
Jul 2012 | Lysozyme | Structure | 1.9 | High-resolution protein structure determination by serial femtosecond crystallography | Boutet et al. | 1 |
Nov 2012 | Thermolysin | Data | 4.0 | Nanoflow electrospinning serial femtosecond crystallography | Sierra et al. | 8 |
Jan 2013 | Cathepsin B | Structure | 2.1 | Natively inhibited Trypsanosoma brucei cathepsin B structure determined by using an X-ray laser | Redecke et al. | 9 |
Apr 2013 | Photosystem II | Structure | 5.7 | Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature | Kern et al. | 10 |
May 2013 | Lysozyme | Structure | 3.2 | Anomalous signal from S atoms in protein crystallographic data from an X-ray free-electron laser | Barends et al. | 11 |
Sept 2013 | Ribosome | Data | <6 | Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser | Demirci et al. | 12 |
Dec 2013 | Photosynthetic Reaction Center | Structure | 3.5 | Structure of a photosynthetic reaction center determined by serial femtosecond crystallography | Johansson et al. | 13 |
Dec 2013 | Serotonin receptor | Structure | 2.8 | Serial femtosecond crystallography of G protein-coupled receptors | Liu et al. | 14 |
Jan 2014 | Lysozyme + Gd | Structure | 2.1 | De novo protein crystal structure determination from XFEL data | Barends et al. | 15 |
This study | Cry3A toxin, isolated crystals and whole cells | Structure | 2.8, 2.9 | 2.9 Å-Resolution protein crystal structure obtained from injecting bacterial cells into an X-ray free-electron laser beam | Sawaya et al. | This study |
19.
Jingling Liao Chun-Xiang Wu Christopher Burlak Sheng Zhang Heather Sahm Mu Wang Zhong-Yin Zhang Kurt W. Vogel Mark Federici Steve M. Riddle R. Jeremy Nichols Dali Liu Mark R. Cookson Todd A. Stone Quyen Q. Hoang 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(11):4055-4060
Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD) (1–5). LRRK2 is a large (2,527-aa) multidomain protein consisting of seven putative domains (2), including a Ras-like GTPase domain called Ras of complex proteins (Roc), followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear how perturbations of these activities result in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild type; therefore, its overactivation might be associated with disease pathogenesis (6).The tandem Roc-COR-Kin arrangement suggests that their activities might be coupled such that the GTPase activity of Roc might modulate the kinase activity. Indeed, several studies have shown that GTP binding to the Roc domain regulates the activity of the Kin domain (7, 8). Moreover, a PD-associated mutation in the Roc domain (R1441C) has been shown to have higher kinase activity (9), thus suggesting that mutations in the Roc domain, also up-regulate kinase activity.Understanding the function of Roc and its mechanism of action is important for understanding the mechanism of PD pathogenesis and therapeutic development. However, because of the lack sufficient quantity of protein samples amendable for detailed investigations, the biochemical properties and enzymatic activities of the Roc domain of LRRK2 are poorly understood.Here, we describe a stably folded construct of human Roc domain that enabled us to investigate quantitatively its biochemical and enzymatic properties. The results revealed that a PD-causing mutation R1441H in the Roc domain renders it less active at hydrolyzing GTP, as well as having higher affinity for GTP, than its wild-type counterpart, thereby increasing the residence time of its GTP-bound “active state,” which is associated with PD pathogenesis (8). 相似文献