Tabelecleucel for allogeneic haematopoietic stem-cell or solid organ transplant recipients with Epstein-Barr virus-positive post-transplant lymphoproliferative disease after failure of rituximab or rituximab and chemotherapy (ALLELE): a phase 3, multicentre, open-label trial.

BACKGROUND: Survival in Epstein-Barr virus (EBV)-positive post-transplant lymphoproliferative disease following haematopoietic stem-cell transplant (HSCT) or solid organ transplant (SOT) is poor after failure of initial therapy, indicating an urgent need for therapies for this ultra-rare disease. With recent EU marketing authorisation, tabelecleucel is the first off-the-shelf, allogeneic, EBV-specific T-cell immunotherapy to receive approval for treatment of relapsed or refractory EBV-positive post-transplant lymphoproliferative disease. We aimed to determine the clinical benefit of tabelecleucel in patients with relapsed or refractory EBV-positive post-transplant lymphoproliferative disease following HSCT or SOT. METHODS: In this global, multicentre, open-label, phase 3 trial, eligible patients (of any age) had biopsy-proven EBV-positive post-transplant lymphoproliferative disease, disease that was relapsed or refractory to rituximab after HSCT and rituximab with or without chemotherapy after SOT, and partially HLA-matched and appropriately HLA-restricted tabelecleucel available. Patients received tabelecleucel administered intravenously at 2 × 106 cells per kg on days 1, 8, and 15 in 35-day cycles and are assessed for up to 5 years for survival post-treatment initiation. The primary endpoint was objective response rate. All patients who received at least one dose of tabelecleucel were included in safety and efficacy analyses. This trial is registered with ClinicalTrials.gov, NCT03394365, and is ongoing. FINDINGS: From June 27, 2018, to Nov 5, 2021, 63 patients were enrolled, of whom 43 (24 [56%] male and 19 [44%] female) were included, 14 had prior HSCT, 29 had SOT. Seven (50%, 95% CI 23-77) of 14 participants in the HSCT group and 15 (52%, 33-71) of 29 participants in the SOT group had an objective response, with a median follow-up of 14·1 months (IQR 5·7-23·9) and 6·0 months (1·8-18·4), respectively. The most common grade 3 or 4 treatment-emergent adverse events were disease progression (in four [29%] of 14 in HSCT and eight [28%] of 29 in SOT) and decreased neutrophil count (in four [29%] of 14 in HSCT and four [14%] of 29 in SOT). Treatment-emergent serious adverse events were reported in 23 (53%) of 43 patients and fatal treatment-emergent adverse events in five (12%); no fatal treatment-emergent adverse event was treatment-related. There were no reports of tumour flare reaction, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, transmission of infectious diseases, marrow rejection, or infusion reactions. No events of graft-versus-host disease or SOT rejection were reported as related to tabelecleucel. INTERPRETATION: Tabelecleucel provides clinical benefit in patients with relapsed or refractory EBV-positive post-transplant lymphoproliferative disease, for whom there are no other approved therapies, without evidence of safety concerns seen with other adoptive T-cell therapies. These data represent a potentially transformative and accessible treatment advance for patients with relapsed or refractory disease with few treatment options. FUNDING: Atara Biotherapeutics.

Two Dof transcription factors promote flavonoid synthesis in kumquat fruit by activating C-glucosyltransferase.

Although DNA binding with one finger (Dof) constitutes a crucial plant-specific family of transcription factors (TFs) that plays important roles in a wide range of biological processes, the molecular mechanisms underlying Dof regulation of flavonoid biosynthesis in plants remain largely unknown. Here, we characterized 28 Dof genes (FhDof1-FhDof28) from the 'Hongkong' kumquat (Fortunella hindsii) cultivar genome. Promoter analysis and transcriptome profiling revealed that four FhDofs - FhDof4, FhDof9, FhDof15, and FhDof16 - may be involved in flavonoid biosynthesis through binding to the flavonoid C-glycosyltransferase (FhCGT) promoter. We cloned homologous genes of four FhDofs, designated as FcDof4, FcDof9, FcDof15, FcDof16, and a homologous gene of FhCGT, designated as FcCGT, from the widely cultivated 'HuaPi' kumquat (F. crassifolia). Quantitative reverse transcription-polymerase chain reaction analysis revealed that FcDof4 and FcDof16 were significantly correlated with FcCGT expression during development stages in the 'HuaPi' fruit (Pearson's correlation coefficient > 0.7) and were localized to the nucleus. Results of yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays indicated that the two FcDofs trigger FcCGT expression by specifically binding to its promoters. Moreover, transient overexpression of FcDof4 and FcDof16 enhances the transcription of structural genes in the flavonoid biosynthetic pathway and increases C-glycosylflavonoid content. Our results provide strong evidence that the TFs FcDof4 and FcDof16 promote flavonoid synthesis in kumquat fruit by activating FcCGT expression.

Teaching basic fiberoptic intubation skills in a simulator: initial learning and skills decay.

PURPOSE: Generally, novices are taught fiberoptic intubation on patients by attending anesthesiologists; however, this approach raises patient safety concerns. Patient safety should improve if novice learners are trained for basic skills on simulators. In this educational study, we assessed the time and number of attempts required to train novices in fiberoptic bronchoscopy and fiberoptic intubation on simulators. Because decay in skills is inevitable, we also assessed fiberoptic bronchoscopy and fiberoptic intubation skill decay and the amount of effort required to regain fiberoptic bronchoscopy skill. METHODS: First, we established attempt- and duration-based quantitative norms for reaching skill proficiency for fiberoptic bronchoscopy and fiberoptic intubation by experienced anesthesiologists (n = 8) and prepared an 11-step checklist and a 5-point global rating scale for assessment. Novice learners (n = 15) were trained to reach the established skill proficiency in a Virtual Reality simulator for fiberoptic bronchoscopy skills and a Human Airway Anatomy Simulator for fiberoptic intubation skills. Two months later, novices were reassessed to determine decay in learned skills and the required time to retrain them to fiberoptic bronchoscopy proficiency level. RESULTS: Proficiency in fiberoptic bronchoscopy skill level was achieved with 11 ± 5 attempts and after 658 ± 351 s. After 2 months without practice, the time taken by the novices to successful fiberoptic bronchoscopy on the Virtual Reality simulator increased from 41 ± 8 to 68 ± 31 s (P = 0.0138). Time and attempts required to retrain them were 424 ± 230 s and 9.1 ± 4.6 attempts, respectively. CONCLUSION: Novices were successfully trained to proficiency skill level. Although fiberoptic bronchoscopy skills started to decay within 2 months, the re-training time was shorter.

Interaction mapping between Saccharomyces cerevisiae Smc5 and SUMO E3 ligase Mms21.

The multisubunit Smc5-Smc6 holocomplex (Smc5/6) plays a critical role in chromosome stability maintenance, DNA replication, homologous recombination, and double-stranded DNA damage repair. Smc5 and Smc6 form the core of the holocomplex, along with six non-SMC elements, for which most functions are not yet understood. Mms21 (Nse2), the relatively well-studied subunit in Smc5/6, contains a SP-like-RING finger motif on the C-terminus and was identified as a SUMO E3 ligase. Deletion of Mms21 is lethal; however, while deficient in DNA damage repair, SUMO ligase mutants remain viable. These functions of Mms21 in Smc5/6 are hard to address without understanding the interaction between Smc5 and Mms21. Previously, we systematically examined the architecture of Saccharomyces cerevisiae Smc5/6 and, using yeast two-hybrid methods, found that Mms21 interacts with the coiled-coil of Smc5. Later, crystallographic studies revealed the molecular arrangement of Mms21 with Smc5/6. For this study, we use a combination of limited proteolysis, mass spectrometry, and N-terminal sequencing to precisely define the interaction region of Smc5 with Mms21. In addition, using isothermal titration calorimetry, we find that Mms21 interacts with Smc5 in a 1:1 ratio with a K(d) of 0.68 µM. This combination of methods would be useful in examining the structure of any large multiprotein complex.

Interplay between the Smc5/6 complex and the Mph1 helicase in recombinational repair.

The evolutionarily conserved Smc5/6 complex is implicated in recombinational repair, but its function in this process has been elusive. Here we report that the budding yeast Smc5/6 complex directly binds to the DNA helicase Mph1. Mph1 and its helicase activity define a replication-associated recombination subpathway. We show that this pathway is toxic when the Smc5/6 complex is defective, because mph1Delta and its helicase mutations suppress multiple defects in mutants of the Smc5/6 complex, including their sensitivity to replication-blocking agents, growth defects, and inefficient chromatid separation, whereas MPH1 overexpression exacerbates some of these defects. We further demonstrate that Mph1 and its helicase activity are largely responsible for the accumulation of potentially deleterious recombination intermediates in mutants of the Smc5/6 complex. We also present evidence that mph1Delta does not alleviate sensitivity to DNA damage or the accumulation of recombination intermediates in cells lacking Sgs1, which is thought to function together with the Smc5/6 complex. Thus, our results reveal a function of the Smc5/6 complex in the Mph1-dependent recombinational subpathway that is distinct from Sgs1. We suggest that the Smc5/6 complex can counteract/modulate a pro-recombinogenic function of Mph1 or facilitate the resolution of recombination structures generated by Mph1.

Structural and functional insights into the roles of the Mms21 subunit of the Smc5/6 complex.

The Smc5/6 complex is an evolutionarily conserved chromosomal ATPase required for cell growth and DNA repair. Its Mms21 subunit supports both functions by docking to the arm region of Smc5 and providing SUMO ligase activity. Here, we report the crystal structure of Mms21 in complex with the Smc5 arm. Our structure revealed two distinct structural and functional domains of the Smc5-bound Mms21: its N-terminal half is dedicated to Smc5 binding by forming a helix bundle with a coiled-coil structure of Smc5; its C-terminal half includes the SUMO ligase domain, which adopts a new type of RING E3 structure. Mutagenesis and structural analyses showed that the Mms21-Smc5 interface is required for cell growth and resistance to DNA damage, while the unique Mms21 RING domain confers specificity to the SUMO E2-E3 interaction. Through structure-based dissection of Mms21 functions, our studies establish a framework for understanding its roles in the Smc5/6 complex.

Purification, crystallization and preliminary X-ray crystallographic studies of the complex between Smc5 and the SUMO E3 ligase Mms21.

Smc5/6, a protein complex that belongs to the structural maintenance of chromosome (SMC) family, plays a key role in DNA replication, sister chromatid recombination and DNA damage repair. The complex contains eight subunits, including a SUMO E3 ligase Mms21 (Nse2). The activity of Mms21 is important for regulation of Smc5/6 in the response to DNA damage. Mms21 and the Mms21-binding region of Smc5 were overexpressed and purified individually in Escherichia coli with a C-terminal LEHHHHHH tag. The Mms21-Smc5 protein complex was crystallized. The diffraction of the crystals was improved greatly by glutaraldehyde treatment. X-ray diffraction data sets were collected to resolutions of 2.3 and 3.9 A from native and selenomethionine-derivative protein crystals, respectively. The crystals belonged to space group C222(1), with unit-cell parameters a = 47.465, b = 97.574, c = 249.215 A for the native crystals.

Architecture of the Smc5/6 Complex of Saccharomyces cerevisiae Reveals a Unique Interaction between the Nse5-6 Subcomplex and the Hinge Regions of Smc5 and Smc6.

The evolutionarily conserved structural maintenance of chromosome (SMC) proteins forms the core structures of three multisubunit complexes as follows: cohesin, condensin, and the Smc5/6 complex. These complexes play crucial roles in different aspects of chromosomal organization, duplication, and segregation. Although the architectures of cohesin and condensin are better understood, that of the more recently identified Smc5/6 complex remains to be elucidated. We have previously shown that the Smc5/6 complex of Saccharomyces cerevisiae contains Smc5, Smc6, and six non-SMC elements (Nse1-6). In this study, we investigated the architecture of the budding yeast Smc5/6 complex employing the yeast two-hybrid assay as well as in vitro biochemical approaches using purified recombinant proteins. These analyses revealed that Smc5 and Smc6 associate with each other at their hinge regions and constitute the backbone of the complex, whereas the Nse1-6 subunits form three distinct subcomplexes/entities that interact with different regions of Smc5 and Smc6. The Nse1, -3, and -4 subunits form a stable subcomplex that binds to the head and the adjacent coiled-coil region of Smc5. Nse2 binds to the middle of the coiled-coil region of Smc5. Nse5 and Nse6 interact with each other and, as a heterodimer, bind to the hinge regions of Smc5 and Smc6. These findings provide new insights into the structures of the Smc5/6 complex and lay the foundation for further investigations into the mechanism of its functions.

Targeting the SUMO E2 conjugating enzyme Ubc9 interaction for anti-cancer drug design.

Sumoylation has been implicated in a variety of cancers, suggesting that sumoylation manipulation could be one approach for regulating tumorgenesis. Ubc9 exerts a central function for the sumoylation pathway, interacting with almost all the partners required for sumoylation. The high-resolution structure available for Ubc9 as well as the recent determination of more interacting partner complex structures makes rational drug design that target Ubc9 possible. Structure-based virtual drug screening has been used increasingly as the first step of drug design to select potential lead templates. This review analyzes all the interfaces between Ubc9 and its binding partners while also highlighting the possible targeting sites on Ubc9 best suited for virtual screening and drug design.

Isolation of cellulolytic enzymes from moldy silage by new culture-independent strategy.

A culture-independent strategy has been developed for investigation of cellulases in moldy silage. By the qualitative differences in the adsorption of cellulases on lingo-cellulosics, a new cellobiohydrolase (CBH) with apparent molecular mass of 194 kDa was isolated and characterized. The entire extracellular proteins of silage were separated by two-dimensional gel electrophoresis, and five potential endoglucanases were identified by activity staining. These results demonstrate the feasibility of direct screening cellulases from environment without microorganism cultivation and this strategy could be expected to facilitate the research of uncultured microorganisms.