In higher plants, the circadian clock orchestrates fundamental processes such as light signaling and the transition to flowering. We isolated mutants of the circadian clock from an Arabidopsis ...thaliana mutagenized reporter line by screening for seedlings with long hypocotyl phenotypes and subsequently assaying for abnormal clock-regulated CAB2::LUC expression. This screen identified five mutant alleles of a clock gene, LUX ARRHYTHMO (LUX), that significantly affect amplitude and robustness of rhythms in both constant white light and dark conditions. In addition, the transition from vegetative to floral development is accelerated and hypocotyl elongation is accentuated in these mutants under light:dark cycles. We genetically mapped the mutations by bulk segregant analysis with high-density oligonucleotide array genotyping to a small putative Myb transcription factor related to other clock components and response regulators in Arabidopsis. The negative arm of the Arabidopsis circadian clock, CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), is repressed in the lux mutants, whereas TIMING OF CAB2 EXPRESSION (TOC1) is activated. We demonstrate that CCA1 and LHY bind to the evening element motif in the LUX promoter, which strongly suggests that these proteins repress LUX expression, as they do TOC1. The data are also consistent with LUX being necessary for activation of CCA1 and LHY expression.
Purpose
Most randomized controlled trials (RCTs) in patients with acute respiratory distress syndrome (ARDS) revealed indeterminate or conflicting study results. We aimed to systematically evaluate ...between-trial heterogeneity in reporting standards and trial outcome.
Methods
A systematic review of RCTs published between 2000 and 2019 was performed including adult ARDS patients receiving lung-protective ventilation. A random-effects meta-regression model was applied to quantify heterogeneity (non-random variability) and to evaluate trial and patient characteristics as sources of heterogeneity.
Results
In total, 67 RCTs were included. The 28-day control-group mortality rate ranged from 10 to 67% with large non-random heterogeneity (
I
2
= 88%,
p
< 0.0001). Reported baseline patient characteristics explained some of the outcome heterogeneity, but only six trials (9%) reported all four independently predictive variables (mean age, mean lung injury score, mean plateau pressure and mean arterial pH). The 28-day control group mortality adjusted for patient characteristics (i.e. the residual heterogeneity) ranged from 18 to 45%. Trials with significant benefit in the primary outcome reported a higher control group mortality than trials with an indeterminate outcome or harm (mean 28-day control group mortality: 44% vs. 28%;
p
= 0.001).
Conclusion
Among ARDS RCTs in the lung-protective ventilation era, there was large variability in the description of baseline characteristics and significant unexplainable heterogeneity in 28-day control group mortality. These findings signify problems with the generalizability of ARDS research and underline the urgent need for standardized reporting of trial and baseline characteristics.
Recombinant adeno-associated virus (rAAV) holds promise as a gene therapy vector for a multitude of genetic disorders such as hemophilia, cystic fibrosis, and the muscular dystrophies. Given the ...variety of applications and tissue types toward which these vectors may be targeted, an understanding of rAAV transduction is crucial for the effective application of therapy. rAAV transduction mechanisms have been the subject of much study, resulting in a body of knowledge relating to events from virus-cell attachment through to vector genome conformation in the target cell nucleus. Instead of utilizing one mechanism in each phase of vector transduction, rAAV appears to employ multiple possible pathways toward transgene expression, in part dependent on rAAV serotype, dose, and target cell type. Once inside the nucleus, the rAAV genome exists in a predominantly episomal form; therefore, nondividing cells tend to be most stably transduced. However, rAAV has a low frequency of integration into the host cell genome, often in or near genes, and can be associated with host genome mutations. This review describes the current understanding of the mechanisms and rate-limiting steps involved in rAAV transduction.
Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is ...normally performed “blind,” meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum.
•A system for robotically targeted neurophysiological recording is demonstrated•The platform keeps the target in focus and corrects the electrode trajectory•The system compensates for micropipette-induced tissue deformations•Targeted recordings were obtained from several fluorescently labeled cells in vivo
Annecchino et al. describe a platform for robotic two-photon targeted patch-clamp recording and demonstrate its functionality by recording from a variety of fluorescently targeted cell types in mouse neocortex and cerebellum.
Recent advances in automated face recognition algorithms have increased the risk that de-identified research MRI scans may be re-identifiable by matching them to identified photographs using face ...recognition. A variety of software exist to de-face (remove faces from) MRI, but their ability to prevent face recognition has never been measured and their image modifications can alter automated brain measurements. In this study, we compared three popular de-facing techniques and introduce our mri_reface technique designed to minimize effects on brain measurements by replacing the face with a population average, rather than removing it. For each technique, we measured 1) how well it prevented automated face recognition (i.e. effects on exceptionally-motivated individuals) and 2) how it altered brain measurements from SPM12, FreeSurfer, and FSL (i.e. effects on the average user of de-identified data). Before de-facing, 97% of scans from a sample of 157 volunteers were correctly matched to photographs using automated face recognition. After de-facing with popular software, 28-38% of scans still retained enough data for successful automated face matching. Our proposed mri_reface had similar performance with the best existing method (fsl_deface) at preventing face recognition (28-30%) and it had the smallest effects on brain measurements in more pipelines than any other, but these differences were modest.
NRG Oncology/RTOG 9802 (ClinicalTrials.gov Identifier: NCT00003375) is a practice-changing study for patients with WHO low-grade glioma (LGG, grade II), as it was the first to demonstrate a survival ...benefit of adjuvant chemoradiotherapy over radiotherapy. This post hoc study sought to determine the prognostic and predictive impact of the WHO-defined molecular subgroups and corresponding molecular alterations within NRG Oncology/RTOG 9802.
mutations were determined by immunohistochemistry and/or deep sequencing. A custom Ion AmpliSeq panel was used for mutation analysis. 1p/19q codeletion and
promoter methylation were determined by copy-number arrays and/or Illumina 450K array, respectively. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Hazard ratios (HRs) were calculated using the Cox proportional hazard model and tested using the log-rank test. Multivariable analyses (MVAs) were performed incorporating treatment and common prognostic factors as covariates.
Of the eligible patients successfully profiled for the WHO-defined molecular groups (n = 106/251), 26 (24%) were
wild type, 43 (41%) were
mutant/non-codeleted, and 37(35%) were
mutant/codeleted. MVAs demonstrated that WHO subgroup was a significant predictor of PFS after adjustment for clinical variables and treatment. Notably, treatment with postradiation chemotherapy (PCV; procarbazine, lomustine (CCNU), and vincristine) was associated with longer PFS (HR, 0.32;
= .003; HR, 0.13;
< .001) and OS (HR, 0.38;
= .013; HR, 0.21;
= .029) in the
mutant/non-codeleted and
mutant/codeleted subgroups, respectively. In contrast, no significant difference in either PFS or OS was observed with the addition of PCV in the
wild-type subgroup.
This study is the first to report the predictive value of the WHO-defined diagnostic classification in a set of uniformly treated patients with LGG in a clinical trial. Importantly, this post hoc analysis supports the notion that patients with
-mutant high-risk LGG regardless of codeletion status receive benefit from the addition of PCV.
We examined current outcomes of unrelated donor allogeneic hematopoietic cell transplantation (HCT) to determine the clinical implications of donor-recipient HLA matching. Adult and pediatric ...patients who had first undergone myeloablative-unrelated bone marrow or peripheral blood HCT for acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome between 1999 and 2011 were included. All had high-resolution typing for HLA-A, -B, -C, and -DRB1. Of the total (n = 8003), cases were 8/8 (n = 5449), 7/8 (n = 2071), or 6/8 (n = 483) matched. HLA mismatch (6-7/8) conferred significantly increased risk for grades II to IV and III to IV acute graft vs host disease (GVHD), chronic GVHD, transplant-related mortality (TRM), and overall mortality compared with HLA-matched cases (8/8). Type (allele/antigen) and locus (HLA-A, -B, -C, and -DRB1) of mismatch were not associated with overall mortality. Among 8/8 matched cases, HLA-DPB1 and -DQB1 mismatch resulted in increased acute GVHD, and HLA-DPB1 mismatch had decreased relapse. Nonpermissive HLA-DPB1 allele mismatch was associated with higher TRM compared with permissive HLA-DPB1 mismatch or HLA-DPB1 match and increased overall mortality compared with permissive HLA-DPB1 mismatch in 8/8 (and 10/10) matched cases. Full matching at HLA-A, -B, -C, and -DRB1 is required for optimal unrelated donor HCT survival, and avoidance of nonpermissive HLA-DPB1 mismatches in otherwise HLA-matched pairs is indicated.
•High-resolution matching for HLA-A, -B, -C, and -DRB1 is required for optimal survival in myeloablative-unrelated donor transplantation.•HLA-DPB1 nonpermissive mismatches should be avoided in otherwise matched transplants to minimize overall mortality.
Pulmonary coagulopathy may play a pathogenetic role in acute respiratory distress syndrome (ARDS), by contributing to alveolocapillary inflammation and increased permeability. Recombinant human ...activated protein C (rh-APC) may inhibit this process and thereby improve patient outcome.
A prospective randomized, saline-controlled, single-blinded clinical trial was performed in the intensive care units of two university hospitals, and patients with ARDS were included within 24 h after meeting inclusion criteria.
A 4-day course of intravenous rh-APC (24 mcg/kg/h) (n = 33) versus saline (n = 38).
The primary outcome parameter was the pulmonary leak index (PLI) of 67Gallium-transferrin as a measure of alveolocapillary permeability and secondary outcomes were disease severity scores and ventilator-free days, among others.
Baseline characteristics were similar; in 87% of patients the PLI was above normal and in 90% mechanical or non-invasive ventilation was instituted at a median lung injury score of 2.5. There was no evidence that Rh-APC treatment affected the PLI or attenuated lung injury and sequential organ failure assessment scores. Mean ventilator-free days amounted to 14 (rh-APC) and 12 days (saline, P = 0.35). 28-day mortality was 6% in rh-APC- and 18% in saline-treated patients (P = 0.12). There was no difference in bleeding events. The study was prematurely discontinued because rh-APC was withdrawn from the market.
There is no evidence that treatment with intravenous rh-APC during 4 days for infectious or inflammatory ARDS ameliorates increased alveolocapillary permeability or the clinical course of ARDS patients. We cannot exclude underpowering.
Nederlands Trial Register ISRCTN 52566874.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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