Conifers Concentrate Large Numbers of NLR Immune Receptor Genes on One Chromosome.

Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant's resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding.

High-density genetic linkage mapping in Sitka spruce advances the integration of genomic resources in conifers.

In species with large and complex genomes such as conifers, dense linkage maps are a useful resource for supporting genome assembly and laying the genomic groundwork at the structural, populational, and functional levels. However, most of the 600+ extant conifer species still lack extensive genotyping resources, which hampers the development of high-density linkage maps. In this study, we developed a linkage map relying on 21,570 single nucleotide polymorphism (SNP) markers in Sitka spruce (Picea sitchensis [Bong.] Carr.), a long-lived conifer from western North America that is widely planted for productive forestry in the British Isles. We used a single-step mapping approach to efficiently combine RAD-seq and genotyping array SNP data for 528 individuals from 2 full-sib families. As expected for spruce taxa, the saturated map contained 12 linkages groups with a total length of 2,142 cM. The positioning of 5,414 unique gene coding sequences allowed us to compare our map with that of other Pinaceae species, which provided evidence for high levels of synteny and gene order conservation in this family. We then developed an integrated map for P. sitchensis and Picea glauca based on 27,052 markers and 11,609 gene sequences. Altogether, these 2 linkage maps, the accompanying catalog of 286,159 SNPs and the genotyping chip developed, herein, open new perspectives for a variety of fundamental and more applied research objectives, such as for the improvement of spruce genome assemblies, or for marker-assisted sustainable management of genetic resources in Sitka spruce and related species.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Patients' roles in governance of learning: Results from a qualitative study of 16 learning healthcare systems.

Patient and family engagement has been identified as key to fulfilling Learning Healthcare Systems' (LHSs') promise as a model for improving clinical care, catalyzing research, and controlling costs. Little is known, however, about the state of patient engagement in the learning mission of these systems or about what governance structures and processes facilitate such engagement. Here, we report on an interview study of 99 patient and employee leaders in 16 systems. We found both variable levels of engagement and broad agreement that shared governance of learning remains a work in progress. We also identified a range of practices that can support or thwart development of an organizational culture conducive to shared governance, including transparency, capacity building, infrastructure investment, leadership, attention to diversity of patient partners, and committee structures. In LHSs with most sophisticated shared governance, both employees and patients contribute to building a democratic learning culture.

Informed consent in pragmatic trials: results from a survey of trials published 2014-2019.

OBJECTIVES: To describe reporting of informed consent in pragmatic trials, justifications for waivers of consent and reporting of alternative approaches to standard written consent. To identify factors associated with (1) not reporting and (2) not obtaining consent. METHODS: Survey of primary trial reports, published 2014-2019, identified using an electronic search filter for pragmatic trials implemented in MEDLINE, and registered in ClinicalTrials.gov. RESULTS: Among 1988 trials, 132 (6.6%) did not include a statement about participant consent, 1691 (85.0%) reported consent had been obtained, 139 (7.0%) reported a waiver and 26 (1.3%) reported consent for one aspect (eg, data collection) but a waiver for another (eg, intervention). Of the 165 trials reporting a waiver, 76 (46.1%) provided a justification. Few (53, 2.9%) explicitly reported use of alternative approaches to consent. In multivariable logistic regression analyses, lower journal impact factor (p=0.001) and cluster randomisation (p<0.0001) were significantly associated with not reporting on consent, while trial recency, cluster randomisation, higher-income country settings, health services research and explicit labelling as pragmatic were significantly associated with not obtaining consent (all p<0.0001). DISCUSSION: Not obtaining consent seems to be increasing and is associated with the use of cluster randomisation and pragmatic aims, but neither cluster randomisation nor pragmatism are currently accepted justifications for waivers of consent. Rather than considering either standard written informed consent or waivers of consent, researchers and research ethics committees could consider alternative consent approaches that may facilitate the conduct of pragmatic trials while preserving patient autonomy and the public's trust in research.

Functional outcomes with handsewn versus stapled anastomoses in the treatment of ultralow rectal cancer.

Adequate oncological outcomes have been demonstrated with rectal resection and handsewn coloanal anastomosis (CAA) in tumours in close proximity to the internal anal sphincter. Our aim was to assess functional differences between handsewn CAA and ultralow stapled anastomosis. Participants were identified from a single-surgeon series. Included participants underwent anorectal physiology testing of anal sphincter function, in addition to completion of several questionnaires: Wexner Incontinence Score (WIS); Birmingham Bowel, Bladder and Urinary Symptom Questionnaire (BBUSQ); Low Anterior Resection Syndrome (LARS) Score; SF36. Non-parametric data compared using the Mann-Whitney U test. 20 participants were included; 11 stapled and 9 handsewn. Mean follow-up was 2.95 ± 1.97 years. The mean LARS score was 21.9 ± 1.97 years in the stapled group versus 29.4 ± 9.57 in the handsewn group (p = 0.133). The Wexner incontinence score was significantly higher in the handsewn group (p = 0.0076), with a mean score of 4.6 ± 3.69 versus 10.9 ± 4.76. The incontinence domain of the BBUSQ was also significantly worse in patients with a handsewn anastomosis (p = 0.001). With the exception of general health (p = 0.035) and social functioning (p = 0.035), which were worse in the handsewn groups, the other six domains of the SF-36 showed no statistical difference between groups. Anorectal physiology scores were not significantly different. Handsewn CAA anastomosis is known to be safe and oncologically feasible. Patient selection should be vigorous, with preoperative counseling regarding the likelihood of incontinence to manage patients' expectations and promote comparable quality of life in the long-term.

Can we correlate pelvic floor dysfunction severity on MR defecography with patient-reported symptom severity?

MR defecography (MRD) is an alternative to conventional defecography (CD) which allows for dynamic visualisation of the pelvic floor. The aim of this study was to assess whether MRI features indicative of pelvic floor dysfunction correlated with patient-reported symptom severity. MR proctograms were matched to a prospectively-maintained functional database. Univariate and multivariate analyses were performed using pre-treatment questionnaire responses to the Birmingham Bowel, Bladder and Urinary Symptom Questionnaire (BBUSQ), Wexner Incontinence Score (WIS), and modified Obstructed Defecation Symptom (ODS) Score. 302 MRI proctograms were performed between January 2012 and April 2015. 170 patients were included. Patients with a rectocele > 2 cm (p = 0.003; OR 5.756) or MRD features suggestive of puborectalis syndrome (p = 0.025; OR 8.602) were more likely to report a higher ODS score on multivariate analysis. Lack of rectal evacuation was negatively associated with an abnormal WIS (p = 0.007; OR 0.228). Age > 50 (p = 0.027, OR 2.204) and a history of pelvic floor surgery (p = 0.042, OR 0.359) were correlated with an abnormal BBUSQ incontinence score. Lack of rectal evacuation (p = 0.027, OR 3.602) was associated with an abnormal BBUSQ constipation score. Age > 50 (p = 0.07, OR 0.156) and the presence of rectoanal intussusception (p = 0.010, OR 0.138) were associated with an abnormal BBUSQ evacuation score. Whilst MRD is a useful tool in aiding multidisciplinary decision making, overall, it is poorly correlated with patient-reported symptom severity, and treatment decisions should not rest solely on results.

CD22 attenuates calcium signaling by potentiating plasma membrane calcium-ATPase activity.

Binding of antigen to the B cell receptor induces a calcium response, which is required for proliferation and antibody production. CD22, a B cell surface protein, inhibits this signal through mechanisms that have been obscure. We report here that CD22 augments calcium efflux after B cell receptor crosslinking. Inhibition of plasma membrane calcium-ATPase (PMCA) attenuated these effects, as did disruption by homologous recombination of the gene encoding PMCA4a and PMCA4b. PMCA coimmunoprecipitated with CD22 in an activation-dependent way. CD22 cytoplasmic tyrosine residues were required for association with PMCA and enhancement of calcium efflux. Moreover, CD22 regulation of efflux and the calcium response required the tyrosine phosphatase SHP-1. Thus, SHP-1 and PMCA provide a mechanism by which CD22, a tissue-specific negative regulator, can affect calcium responses.

Effects of substrates (methyl isocyanide, C2H2) and inhibitor (CO) on resting-state wild-type and NifV(-)Klebsiella pneumoniae MoFe proteins.

We report the use of electron nuclear double resonance (ENDOR) spectroscopy to examine how the metal sites in the FeMo-cofactor cluster of the resting nitrogenase MoFe protein respond to addition of the substrates acetylene and methyl isocyanide and the inhibitor carbon monoxide. 1H, 57Fe and 95Mo ENDOR measurements were performed on the wild-type and the NifV(-)proteins from Klebsiella pneumoniae. Among the molecules tested, only the addition of acetylene to either protein induced widespread changes in the 57Fe ENDOR spectra. Acetylene also induced increases in intensity from unresolved protons in the proton ENDOR spectra. Thus we conclude that acetylene may bind to the resting-state MoFe protein to perturb the FeMo-cofactor environment. On the other hand, the present results show that methyl isocyanide and carbon monoxide do not substantially alter the FeMo cofactor's geometric and electronic structures. We interpret this as lack of interaction between those two molecules and the FeMo cofactor in the resting state MoFe protein. Thus, although it is generally accepted that substrates or inhibitors bind to the FeMo-cofactor only under turnover condition, this work provides evidence that at least one substrate can perturb the active site of nitrogenase under non-catalytic conditions.

Sialic acid binding domains of CD22 are required for negative regulation of B cell receptor signaling.

CD22, a negative regulator of B cell antigen receptor signaling, binds glycoconjugates terminating in alpha2, 6 sialic acid. The physiological ligand(s) for CD22 remain unknown. We asked whether the sialic acid binding domains are necessary for CD22 to function as a negative regulator. We generated two mutants that lack sialic acid binding activity and expressed them in a novel CD22(-/-) murine B cell line. Anti-IgM activated B cells expressing either CD22 mutant had greater Ca(2+) responses than cells expressing wild-type CD22. Each variant also had reduced CD22 tyrosine phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1 association. These data suggest that the alpha2, 6 sialic acid ligand binding activity of CD22 is critical for its negative regulatory functions.

Combined Clozapine and Electroconvulsive Therapy.

We reviewed consecutive patients (n = 12) at McLean Hospital from 1990 through 1991 treated with the combination of the atypical antipsychotic agent clozapine and electroconvulsive therapy (ECT). There were no adverse effects. Three patients had a marked clinical improvement, one a moderate response, four a minimal response, two minimal to no response, and two no response. Using daily doses of up to 550 mg clozapine, this combination appears to be safe, and may be useful in some patients with treatment-refractory psychosis.