29 research outputs found
PCR-based gene synthesis to produce recombinant proteins for crystallization
- Publication venue
- BioMed Central
- Publication date
- 01/01/2008
- Field of study
<p>Abstract</p> <p>Background</p> <p>Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves.</p> <p>Results</p> <p>A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille) domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA). The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to <it>in vivo </it>homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in <it>E. coli </it>and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis.</p> <p>Conclusion</p> <p>We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.</p
Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.
- Author
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- Ab Rahman AS
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- Publication venue
- 'Oxford University Press (OUP)'
- Publication date
- 01/01/2016
- Field of study
BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700
Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study
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- 'American Society of Clinical Oncology (ASCO)'
- Publication date
- 01/01/2021
- Field of study
PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks
Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.
- Author
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- Zullo A.
- Publication venue
- J Clin Oncol
- Publication date
- 01/01/2020
- Field of study
PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks
Enhancing Abiotic Stress Tolerance in Cereals Through Breeding and Transgenic Interventions
- Author
- Al Shahrour
- Ali
- Ali
- Alves da Costa
- Andrew
- Araus
- Araus
- Ashraf
- Ashraf
- Azooz
- Babu
- Baheri
- Bahieldin
- Baier
- Balfourier
- Barnabás
- Bartels
- Baum
- Bennet
- Bernier
- Bhalla
- Bhat
- Bidinger
- Bidinger
- Bidinger
- Birch
- Blum
- Blum
- Borrell
- Borrell
- Borrell
- Bortiri
- Boyer
- Brini
- Buchanan
- Byrne
- Byrt
- Bäanziger
- Campos
- Caniato
- Capell
- Carlson
- Carvalho
- Carver
- Castiglioni
- Cattivelli
- Ceccarelli
- Chandrababu
- Chaves
- Chen
- Chimenti
- Chopra
- Colmer
- Colmer
- Condon
- Conway
- Cuadrado
- Das-Chatterjee
- Dashti
- Datta
- Davenport
- de Costa
- de Dorlot
- Delhaize
- Denby
- Diab
- Diener
- Ding
- Donaldson
- Dubcovsky
- Dubouzet
- Dugas
- Dwivedi
- Díaz De León
- El-Hendawy
- Eticha
- Farshadfar
- Ferrio
- Flowers
- Flowers
- Flowers
- Flowers
- Fontecha
- Forster
- Forster
- Foulkes
- Foy
- Frova
- Fu
- Fujii
- Fukuda
- Furukawa
- Gan
- Garg
- Garthwaite
- Genc
- Giuliani
- Gorham
- Gororo
- Gregersen
- Gregorio
- Guo
- Harris
- Hassnian
- Hirasawa
- Hirayama
- Hoekenga
- Horie
- Hoshida
- Hossain
- Hu
- Hu
- Huang
- Huang
- Huang
- Hui
- ICRISAT
- ICRISAT
- IRGSP
- Ishida
- Ismail
- Ito
- James
- Jiang
- Jithesh
- Jones
- Kamara
- Kamoshita
- Karaba
- Kasuga
- Kebede
- Khan
- Khan
- King
- King
- Kirigwi
- Kishitani
- Kochian
- Kochian
- Koyama
- Krishnamurthy
- Krishnamurthy
- Kubo
- Kumar
- Kumar
- Kumar
- Kumari
- Kusaka
- Lafitte
- Lanceras
- Landi
- Lang
- Lee
- Li
- Li
- Li
- Li
- Ligaba
- Lin
- Lin
- Lindsay
- Liu
- Liu
- Liu
- Llugany
- Long
- Lorence
- Lu
- Luo
- Ma
- Ma
- Ma
- Ma
- Ma
- Ma
- Magalhaes
- Magalhaes
- Mahalakshmi
- Majee
- Malysheva-Otto
- Manickavelu
- Manneh
- Mano
- Mano
- Mariano
- Mathur
- McGuire
- McKendry
- Meier
- Mifflin
- Milford
- Miller
- Minella
- Miranda
- Mohanty
- Molnár
- Monneveux
- Monneveux
- Monneveux
- Morison
- Mullan
- Munns
- Munns
- Munns
- Munns
- Munns
- Nakashima
- Nasir
- Natrajan
- Nawrot
- Nelson
- Nevo
- Nguyen
- Nguyen
- Nguyen
- Nguyen
- Ninamango-Cárdenas
- Njau
- Noaman
- Oh
- Ohta
- Ortiz
- Ortiz
- Ouk
- Palatnik
- Parry
- Passioura
- Paterson
- Peleg
- Pellegrineschi
- Peng
- Pennisi
- Ping
- Piñeros
- Polle
- Prashanth
- Price
- Price
- Price
- Price
- Pritchard
- Qin
- Quan
- Raman
- Raman
- Raman
- Rampino
- Rebetzke
- Ren
- Rengasamy
- Reynolds
- Reynolds
- Ribaut
- Ribaut
- Ribaut
- Richards
- Riede
- Rivero
- Robin
- Rockström
- Rooney
- Rothan
- Roy
- Roy
- Saijo
- Salzman
- Samac
- Sanchez
- Sanguineti
- Sanguineti
- Sari-Gorla
- Sasaki
- Schachtman
- Schnyder
- Seki
- Shahram-Moammady
- Sharma
- Shen
- Shi
- Shirasawa
- Shou
- Shrawat
- Shylaraj
- Shylaraj
- Shylaraj
- Sibov
- Sivamani
- Slafer
- Snape
- Steele
- Stodart
- Subudhi
- Sunkar
- Suo
- Suprunova
- Swindell
- Sánchez-Urdaneta
- Tajbakhsh
- Takeda
- Tambussi
- Tanaka
- Tang
- Teulat
- Teulat
- Thalji
- Thomson
- Tondelli
- Tsuji
- Tuberosa
- Tuberosa
- Tuberosa
- Tuberosa
- Tuinstra
- Umezawa
- Valliyodan
- Vargas
- Varshney
- Venuprasad
- Verma
- Vinod
- Wang
- Wang
- Wang
- Wang
- Wang
- Wang
- Wang
- WARDA
- Welcker
- Wood
- Wu
- Wu
- Wu
- Xiang
- Xiao
- Xiao
- Xiong
- Xu
- Xu
- Xu
- Xu
- Xue
- Xue
- Xue
- Xue
- Xue
- Xue
- Yadav
- Yadav
- Yadav
- Yadav
- Yang
- Yang
- Yue
- Yue
- Zaharieva
- Zaidi
- Zeven
- Zhai
- Zhang
- Zhang
- Zhao
- Zhao
- Zhao
- Zheng
- Zheng
- Zhou
- Zhou
- Zhu
- Zhu
- Zimmermann
- Zou
- Publication venue
- 'Wiley'
- Publication date
- 01/01/2010
- Field of study
The success of plant breeding in the 20th century led to new cultivars that, to date, have provided enough food for an increasing world population (Conway and Toenniessen 1999; Mifflin 2000). The results of the Green Revolution-led in the 1960s by Henry M. Beachell andNormanE. Dotlaug—resulted in a dramatic increase in rice and wheal grain yields (Milford and Runge 2007; Ortiz et al. 2007). However, abiotic stresses and climate change are becoming increasingly serious threats to crop production worldwide at a time when food staple supply will need to be significantly higher to meet the demand of the growing human population. Water scarcity (Rockstrom et al. 2007], salinity (Rengasamy 2006). and low soil fertility (Sanchez and Swaminathan 2005) rank among the moat important abiotic stresses worldwide. Similarly, increased climatic disturbances due to global warming are causing the major stresses that necessitate crop improvements to safeguard grain supply, particularly in the developing world (Kumar 2006). Hence, genetic enhancement of cereal crops with respect to abiotic stress tolerance will be essential far ensuring grain yields in water-limited, increasingly hotter agricultural zones, particularly If these conditions combine with poor and saline soils, conditions that prevail in parts of the developing world. Crop breeding for adaptation to abiotic stress-prone environments remains a challenging task, not least because of the complexity of the stress-adaptive mechanisms in plants and particularly cereal crops, which are the staple of most of the world's population (Reynolds et al. 2005}
31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two
- Author
- Abassi Yama
- Abelin Jennifer G
- Abolhalaj Milad
- Abraham Tara S
- Adam Ammar
- Adams David
- Adams Homer
- Adams Sarah F
- Adamus Tomasz
- Addepalli Murali
- Addepalli Murali
- Adjei Alex A
- Agapova Larissa
- Agarwala Sanjiv
- Ager Casey
- Aggarwal Charu
- Agnello Giulia
- Agudo Judith
- Aguilar Ethan G
- Aguilera Todd
- Ahamadi Malidi
- Ahn Yong-Oon
- Ahrens Eric T
- Aizer Ayal
- Ajina Reham
- Akerley Wallace
- Al-Muftah Mariam
- Albershardt Tina C
- Albershardt Tina C
- Albrekt Ann-Sofie
- Alekar Shilpa
- Alemany Ramon
- Alemany Ramon
- Alexander Brian
- Allbritton Omaira
- Allen Clint T
- Alley Evan W
- Allison James
- Allison James
- Allison James
- Alston Tesha
- Alt Jürgen
- Alters Susan
- Amatulli Michael
- Anand Snjezana
- Anand Snjezana
- Anand Snjezana
- Anderson Clark
- Anderson Mark
- Andersson Bengt
- Andre Pascale
- Andtbacka Robert H I
- Andtbacka Robert H I
- Angiuoli Sam
- Ansari Tameem
- Anthony Scott
- Antonarakis Emmanuel S
- Antonia Scott J
- Anwar Firoz
- Aquino-Michaels Keston
- Archer Jacob F
- Arina Ainhoa
- Armand Philippe
- Armenta Paul
- Armet Caroline M
- Arnoux Thomas
- Ascarateil Stephane
- Askmyr David
- Atkins Michael
- Atkins Michael B
- Atkinson Victoria
- Au Katrina
- Autio Karen A
- Awad Mark
- Bagarazzi Mark
- Bai Dina
- Baia Gilson
- Baird Jason
- Bajaj Anshika
- Balatoni Timea
- Balboni Tracy
- Balch Leslie
- Bang Andrew
- Bao Riyue
- Bao Yangyi
- Bao Yangyi
- Barakat David
- Barberi Theresa
- Barker Christopher A
- Barnea Eytan
- Barras David
- Bartkowiak Todd
- Bartlett David
- Bartlett David
- Bartlett David
- Bartlett David
- Barton Sunjay M
- Barve Minal
- Bauer Todd
- Bauer Todd W
- Bauman Julie
- Baumgaertner Petra
- Bauml Joshua
- Beceren-Braun Figen
- Beck Kristen
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- Beckers Johannes
- Beckett Michael A
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- Bedognetti Davide
- Bedognetti Davide
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- Bell Charles J M
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- Beltran Pedro
- Bender Lewis H
- Bender Steven
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- Berzofsky Jay A
- Bhardwaj Nina
- Bian Zhen
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- Biediger Ronald J
- Bifulco Carlo
- Bigley Alison
- Bingham Patrick
- Biniszkiewicz Detlev M
- Bischoff Helge
- Blake Zoe
- Blake Zoe
- Blakely Collin
- Blazar Bruce R
- Blogowski Wojciech
- Bloom Anja C
- Boehm Jesse
- Bokovanov Vladimir
- Bommareddy Praveen K
- Bommareddy Praveen K
- Bommareddy Praveen K
- Bommareddy Praveen K
- Booth Jamie
- Bornschlegl Svetlana
- Bose Nandita
- Bossenmaier Birgit
- Boughorbel Sabri
- Boyer Jean
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- Branstetter Daniel
- Brech Dorothee
- Brenin David
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- Brockstedt Dirk G
- Brody Joshua
- Bronson Roderick
- Brooks Alan
- Brooks Alan D
- Broucek Joseph
- Brown Alice
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- Butterfield Lisa H
- Butterfield Lisa H
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- Caplazi Patrick
- Cappuccini Federica
- Carcaboso Angel M
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- Carpi Sara
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- Caux Christophe
- Cebon Jonathan S
- Ceccarelli Michele
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- Cerullo Vincenzo
- Cervera-Carrascón Víctor
- Cesareni Gianni
- Cha John
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- Chan Chanty
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- Chan Leo
- Chan Timothy A
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- Chang Thomas
- Chanuc Fabien
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- Chappel Scott C
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- Corrales Leticia P
- Coukos George
- Coukos George
- Coussens Lisa M
- Coussens Lisa M
- Craggs Graham
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- Cronstein Bruce N
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- DiCostanzo AriCeli
- Diede Scott J
- Dietz Allan B
- Dillhoff Mary
- Dillon Patrick
- Dilworth Ryan
- Dimberg Anna
- Ding Yueyun
- Doberstein Stephen K
- Dobkin Julie
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- Doman Thompson
- Dominguez George
- Donahue Renee
- Donahue Renee
- Dorsey Frank C
- Dovedi Simon
- Downs-Canner Stephanie
- Drake Charles
- Drake Charles G
- Drake Charles G
- Driessens Gregory
- Driscoll Kyla
- Druker Brian J
- Dubensky Thomas W
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- Fesenkova Valentyna
- Field Jessica J
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- Fisher Terry
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- Fuertes Marraco Silvia A
- Fugle Caroline W
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- Gajewski Thomas F
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- Galeassi Nadejda
- Gamble Alicia
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- Gameiro Sofia
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- Gartrell Robyn
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- Gartrell Robyn
- Gasmi Billel
- Gastineau Dennis A
- Gaughan Elizabeth
- Gauthier Audrey
- Gauthier Kelsey S
- Geissler Michael
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- Grossenbacher Steven K
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- Gupta Ravi
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- Gupta Sumati
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- Gutierrez Andres A
- Guttridge Denis
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- Hailemichael Yared
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- Hale Diane F
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- Hendrickx Wouter
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- Hodge James
- Hodge James
- Hodge James W
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- Hodi F. S
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- Hofmeister Robert
- Holland Pamela M
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- Hollingsworth Robert E
- Hollingsworth Robert E
- Holmgaard Rikke
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- Huang Yuanhiu
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- Huelsmann Erica
- Huffman Austin P
- Humeau Laurent
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- Kumar Sushil
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- Kung Andrew L
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- Kuo Peiwen
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- Laderas Ted
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- Lin Jing
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- Lindstedt Malin
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- Liu Zhuqing
- Lo Kin-Ming
- Loibner Hans
- Long Georgina V
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- Loskog Angelica
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- Lu Yan
- Lu Yan
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- Maurer Matthew
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- Zheng Yi
- Zhong Hong
- Zhou Li
- Zhou Xiangjun
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- Zloza Andrew
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- Zuba-Surma Ewa
- Zubkova Olga
- Zureikat Amer
- Publication venue
- 'Springer Science and Business Media LLC'
- Publication date
- 01/01/2016
- Field of study
Background
The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd.
Methods
We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background.
Results
First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001).
Conclusions
In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival
Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducens
- Publication venue
- International Union of Crystallography
- Publication date
- Field of study
The proliferating cell nuclear antigen (PCNA) from a novel hyperthermophilic archaeon Thermococcus thioreducens has been crystallized, and diffraction data have been collected to 1.86 Å
1040-H Coupling DNA barcoding and multi-peptide screening: unveiling T cell receptor and antigenic peptide relationships in complex samples
- Publication venue
- BMJ Publishing Group
- Publication date
- 01/11/2023
- Field of study
PCR-based gene synthesis to produce recombinant proteins for crystallization-1
- Publication venue
- Publication date
- Field of study
Ted initially to sequence analysis of the synthesized product to detect nucleotide errors. Error correction can be conducted in two ways using oligonucleotide primers (30–35 nucleotides long) that are designed to include the correcting nucleotide (when needed) at the midpoint. First, mutagenic primers are targeted exclusively against the assembled synthetic gene (route A). Two, three and four pairs of primers are required to correct one, two and three point mutation (ptm) sites respectively. DNA fragments are amplified by two or all primer pairs F1-R1, F2-R2, F3-R3 and F1-R4 in separate reactions. The terminal primers have overlapping homologous regions with that of the targeted plasmid vector. Reaction products are mixed for transformation into an appropriate cell host. The second approach involves the amplification of the plasmid vector (routes B-D). To remove 3 point mutations, two correcting primers, reverse-complement of each other, are designed at each mutation site, with the correcting nucleotide being at the midpoint of each primer. DNA fragments are amplified by PCR using primer pairs F1-R1, F2-R2 and F3-R3 respectively in 3 separate reactions (D). Two pairs of primers are similarly used for 2 point mutations involving only 2 separate reactions (C). Single site error correction requires a non-mutagenic primer pair corresponding to a sequence in the vector backbone in addition to the correcting primer set such that 2 fragments are generated (as if 2 corrections were being made). Products of the correcting reactions are retransformed into competent cells for plasmid isolation and sequencing. Upon verification of error free clones, the plasmids are then transformed into an appropriate host cell for protein expression.<p><b>Copyright information:</b></p><p>Taken from "PCR-based gene synthesis to produce recombinant proteins for crystallization"</p><p>http://www.biomedcentral.com/1472-6750/8/44</p><p>BMC Biotechnology 2008;8():44-44.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2408586.</p><p></p