High Amount of Transcription Factor IRF8 Engages AP1-IRF Composite Elements in Enhancers to Direct Type 1 Conventional Dendritic Cell Identity.

Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.

A Noncanonical CD56dimCD16dim/- NK Cell Subset Indicative of Prior Cytotoxic Activity Is Elevated in Patients with Autoantibody-Mediated Neurologic Diseases.

Neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein Ab disease, and autoimmune myasthenia gravis (MG) are autoantibody-mediated neurologic conditions where autoantibodies can induce Ab-dependent cellular cytotoxicity (ADCC), a NK cell-mediated effector function. However, whether ADCC is a pathogenic mechanism in patients with these conditions has not been confirmed. We sought to characterize circulatory NK cells using functional assays, phenotyping, and transcriptomics to elucidate their role in pathology. NK cells from NMOSD patients and MG patients with elevated disease burden exhibited reduced ADCC and CD56dimCD16hi NK cells, along with an elevated frequency of CD56dimCD16dim/- NK cells. We determined that ADCC induces a similar phenotypic shift in vitro. Bulk RNA sequencing distinguished the CD56dimCD16dim/- population from the canonical CD56dimCD16hi cytotoxic and CD56hiCD16- immunomodulatory subsets, as well as CD56hiCD16+ NK cells. Multiparameter immunophenotyping of NK cell markers, functional proteins, and receptors similarly showed that the CD56dimCD16dim/- subset exhibits a unique profile while still maintaining expression of characteristic NK markers CD56, CD94, and NKp44. Notably, expression of perforin and granzyme is reduced in comparison with CD56dimCD16hi NK cells. Moreover, they exhibit elevated trogocytosis capability, HLA-DR expression, and many chemokine receptors, including CCR7. In contrast with NMOSD and MG, myelin oligodendrocyte glycoprotein Ab disease NK cells did not exhibit functional, phenotypic, or transcriptomic perturbations. In summary, CD56dimCD16dim/- NK cells are a distinct peripheral blood immune cell population in humans elevated upon prior cytotoxic activity by the CD56dimCD16hi NK cell subset. The elevation of this subset in NMOSD and MG patients suggests prior ADCC activity.

Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin.

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

Bcl6, Irf2, and Notch2 promote nonclassical monocyte development.

Ly6Clo monocytes are a myeloid subset that specializes in the surveillance of vascular endothelium. Ly6Clo monocytes have been shown to derive from Ly6Chi monocytes. NOTCH2 signaling has been implicated as a trigger for Ly6Clo monocyte development, but the basis for this effect is unclear. Here, we examined the impact of NOTCH2 signaling of myeloid progenitors on the development of Ly6Clo monocytes in vitro. NOTCH2 signaling induced by delta-like ligand 1 (DLL1) efficiently induced the transition of Ly6Chi TREML4- monocytes into Ly6Clo TREML4+ monocytes. We further identified two additional transcriptional requirements for development of Ly6Clo monocytes. Deletion of BCL6 from myeloid progenitors abrogated development of Ly6Clo monocytes. IRF2 was also required for Ly6Clo monocyte development in a cell-intrinsic manner. DLL1-induced in vitro transition into Ly6Clo TREML4+ monocytes required IRF2 but unexpectedly could occur in the absence of NUR77 or BCL6. These results imply a transcriptional hierarchy for these factors in controlling Ly6Clo monocyte development.

Identification of genetic variants of the industrial yeast Komagataella phaffii (Pichia pastoris) that contribute to increased yields of secreted heterologous proteins.

The yeast Komagataella phaffii (formerly called Pichia pastoris) is used widely as a host for secretion of heterologous proteins, but only a few isolates of this species exist and all the commonly used expression systems are derived from a single genetic background, CBS7435 (NRRL Y-11430). We hypothesized that other genetic backgrounds could harbor variants that affect yields of secreted proteins. We crossed CBS7435 with 2 other K. phaffii isolates and mapped quantitative trait loci (QTLs) for secretion of a heterologous protein, ß-glucosidase, by sequencing individual segregant genomes. A major QTL mapped to a frameshift mutation in the mannosyltransferase gene HOC1, which gives CBS7435 a weaker cell wall and higher protein secretion than the other isolates. Inactivation of HOC1 in the other isolates doubled ß-glucosidase secretion. A second QTL mapped to an amino acid substitution in IRA1 that tripled ß-glucosidase secretion in 1-week batch cultures but reduced cell viability, and its effects are specific to this heterologous protein. Our results demonstrate that QTL analysis is a powerful method for dissecting the basis of biotechnological traits in nonconventional yeasts, and a route to improving their industrial performance.

Airlift bioreactor-based strategies for prolonged semi-continuous cultivation of edible Agaricomycetes.

Submerged cultivation of edible filamentous fungi (Agaricomycetes) in bioreactors enables maximum mass transfer of nutrients and has the potential to increase the volumetric productivity of fungal biomass compared to solid state cultivation. These aspects are paramount if one wants to increase the range of bioactives (e.g. glucans) in convenient time frames. In this study, Trametes versicolor (M9911) outperformed four other Agaricomycetes tested strains (during batch cultivations in an airlift bioreactor). This strain was therefore further tested in semi-continuous cultivation. Continuous and semi-continuous cultivations (driven by the dilution rate, D) are the preferred bioprocess strategies for biomass production. We examined the semi-continuous cultivation of T. versicolor at dilution rates between 0.02 and 0.1 h-1. A maximum volumetric productivity of 0.87 g/L/h was obtained with a D of 0.1 h-1 but with a lower total biomass production (cell dry weight, CDW 8.7 g/L) than the one obtained at lower dilution rates (12.3 g/L at D of 0.04 and vs 13.4 g/L, at a D of 0.02 h-1). However, growth at a D of 0.1 h-1 resulted in a very short fermentation (18 h) which terminated due to washout (the specific D exceeded the maximum growth rate of the fungal biomass). At a D of 0.04 h-1, a CDW of 12.3 g/L was achieved without compromising the total residence time (184 h) of the fermentation. While the D of 0.04 h-1 and 0.07 h-1 achieved comparable volumetric productivities (0.5 g/L/h), the total duration of the fermentation at D of 0.07 h-1 was only 85 h. The highest glucan content of cells (27.8 as percentage of CDW) was obtained at a D of 0.07 h-1, while the lowest glucan content was observed in T. versicolor cells grown at a D of 0.02 h-1. KEY POINTS: • The highest reported volumetric productivity for fungal biomass was 0.87 g/L/h. • Semi-continuous fermentation at D of 0.02 h-1 resulted in 13.4 g/L of fungal biomass. • Semi-continuous fermentation at D of 0.07 h-1 resulted in fungal biomass with 28% of total glucans.

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

Individual myasthenia gravis autoantibody clones can efficiently mediate multiple mechanisms of pathology.

Serum autoantibodies targeting the nicotinic acetylcholine receptor (AChR) in patients with autoimmune myasthenia gravis (MG) can mediate pathology via three distinct molecular mechanisms: complement activation, receptor blockade, and antigenic modulation. However, it is unclear whether multi-pathogenicity is mediated by individual or multiple autoantibody clones. Using an unbiased B cell culture screening approach, we generated a library of 11 human-derived AChR-specific recombinant monoclonal autoantibodies (mAb) and assessed their binding properties and pathogenic profiles using specialized cell-based assays. Five mAbs activated complement, three blocked α-bungarotoxin binding to the receptor, and seven induced antigenic modulation. Furthermore, two clonally related mAbs derived from one patient were each highly efficient at more than one of these mechanisms, demonstrating that pathogenic mechanisms are not mutually exclusive at the monoclonal level. Using novel Jurkat cell lines that individually express each monomeric AChR subunit (α2ßδε), these two mAbs with multi-pathogenic capacity were determined to exclusively bind the α-subunit of AChR, demonstrating an association between mAb specificity and pathogenic capacity. These findings provide new insight into the immunopathology of MG, demonstrating that single autoreactive clones can efficiently mediate multiple modes of pathology. Current therapeutic approaches targeting only one autoantibody-mediated pathogenic mechanism may be evaded by autoantibodies with multifaceted capacity.

Assessment of association between lower ureteric excision technique and oncological outcomes for upper urinary tract urothelial carcinoma: retrospective analysis from the Scottish Renal Cancer Consortium.

PURPOSE: Nephroureterectomy(NU) remains the gold-standard surgical option for the management of upper urinary tract urothelial carcinoma(UTUC). Controversy exists regarding the optimal excision technique of the lower ureter. We sought to compare post-UTUC bladder tumour recurrence across the Scottish Renal Cancer Consortium(SRCC). METHODS: Patients who underwent NU for UTUC across the SRCC 2012-2019 were identified. The impact of lower-end surgical technique along with T-stage, N-stage, tumour location and focality, positive surgical margin, pre-NU ureteroscopy, upper-end technique and adjuvant mitomycin C administration were assessed by Kaplan-Meier and Cox-regression. The primary outcome was intra-vesical recurrence-free survival (B-RFS). RESULTS: In 402 patients, the median follow-up was 29 months. The lower ureter was managed by open transvesical excision in 90 individuals, transurethral and laparoscopic dissection in 76, laparoscopic or open extra-vesical excision in 31 and 42 respectively, and transurethral dissection and pluck in 163. 114(28.4%) patients had a bladder recurrence during follow-up. There was no difference in B-RFS between lower-end techniques by Kaplan-Meier (p = 0.94). When all factors were taken into account by adjusted Cox-regression, preceding ureteroscopy (HR 2.65, p = 0.001), lower ureteric tumour location (HR 2.16, p = 0.02), previous bladder cancer (HR 1.75, p = 0.01) and male gender (HR 1.61, p = 0.03) were associated with B-RFS. CONCLUSION: These data suggest in appropriately selected patients, lower ureteric management technique does not affect B-RFS. Along with lower ureteric tumour location, male gender and previous bladder cancer, preceding ureteroscopy was associated with a higher recurrence rate following NU, and the indication for this should be carefully considered.

Bcl6-Independent In Vivo Development of Functional Type 1 Classical Dendritic Cells Supporting Tumor Rejection.

The transcriptional repressor Bcl6 has been reported as required for development of a subset of classical dendritic cell (cDCs) called cDC1, which is responsible for cross-presentation. However, mechanisms and in vivo functional analysis have been lacking. We generated a system for conditional deletion of Bcl6 in mouse cDCs. We confirmed the reported in vitro requirement for Bcl6 in cDC1 development and the general role for Bcl6 in cDC development in competitive settings. However, deletion of Bcl6 did not abrogate the in vivo development of cDC1. Instead, Bcl6 deficiency caused only a selective reduction in CD8α expression by cDC1 without affecting XCR1 or CD24 expression. Normal cDC1 development was confirmed in Bcl6cKO mice by development of XCR1+ Zbtb46-GFP+ cDC1 by rejection of syngeneic tumors and by priming of tumor-specific CD8 T cells. In summary, Bcl6 regulates a subset of cDC1-specific markers and is required in vitro but not in vivo for cDC1 development.

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.

ß-oxidation-polyhydroxyalkanoates synthesis relationship in Pseudomonas putida KT2440 revisited.

Pseudomonas putida KT2440 is a well-known model organism for the medium-chain-length (mcl) polyhydroxyalkanoate (PHA) accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the ß-oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC, are annotated in P. putida KT2440. To investigate the relationship of fatty acids-PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in wild type (WT) and mutants under different growth conditions were analysed. PhaJ4 was the main monomer supplier for PHA synthesis with FA as sole carbon and energy source, with preference towards C8 and C10 substrate, whereas PhaJ1 showed preference for the C6 substrate. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7% of the cell dry weight (CDW). The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing a further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogenases PedE and PedH as potential monomer suppliers, but when these were deleted, the PHA level remained at 2.2-14.8% CDW depending on the fatty acid used and whether nitrogen limitation was applied. Therefore, it is likely that some other non-specific dehydrogenases supply monomers for PHA synthesis, demonstrating the redundancy of PHA metabolism. KEY POINTS: • ß-oxidation intermediates are converted to PHA monomers by hydratases PhaJ1, PhaJ4 and MaoC in Pseudomonas putida KT2440. • When these are deleted, the PHA level decreases, but it is not abolished. • PHA non-specific enzyme(s) also contributes to PHA metabolism in KT2440.

Pre-treatment differential correlation of gene expression and response to topical steroids in eosinophilic esophagitis.

Few predictors of response to topical corticosteroid (tCS) treatment have been identified in eosinophilic esophagitis (EoE). We aimed to determine whether baseline gene expression predicts histologic response to tCS treatment for EoE. We analyzed prospectively collected samples from incident EoE cases who were treated with tCS for 8 weeks in a development cohort (prospective study) or in an independent validation cohort (clinical trial). Whole transcriptome RNA expression was determined from a baseline (pre-treatment) RNA-later preserved esophageal biopsy. Baseline expression was compared between histologic responders (<15 eos/hpf) and non-responders (≥15 eos/hpf), and differential correlation was used to assess baseline gene expression by response status. In 87 EoE cases analyzed in the development set, there were no differentially expressed genes associated with treatment response (at false discovery rate = 0.1). However, differential correlation identified a module of 22 genes with statistically significantly high pairwise correlation in non-responders (mean correlation coefficient = 0.7) compared to low correlation in responders (coefficient = 0.3). When this 22-gene module was applied to the 89 EoE cases in the independent cohort, it was not validated to predict tCS response at the 15 eos/hpf threshold (mean correlation coefficient = 0.32 in responders and 0.25 in nonresponders). Exploration of other thresholds also did not validate any modules. Though we identified a 22 gene differential correlation module measured pre-treatment that was strongly associated with subsequent histologic response to tCS in EoE, this was not validated in an independent population. Alternative methods to predict steroid response should be explored.

Experimentally determined strengths of favorable and unfavorable interactions of amide atoms involved in protein self-assembly in water.

Folding and other protein self-assembly processes are driven by favorable interactions between O, N, and C unified atoms of the polypeptide backbone and side chains. These processes are perturbed by solutes that interact with these atoms differently than water does. Amide NH···O=C hydrogen bonding and various π-system interactions have been better characterized structurally or by simulations than experimentally in water, and unfavorable interactions are relatively uncharacterized. To address this situation, we previously quantified interactions of alkyl ureas with amide and aromatic compounds, relative to interactions with water. Analysis yielded strengths of interaction of each alkylurea with unit areas of different hybridization states of unified O, N, and C atoms of amide and aromatic compounds. Here, by osmometry, we quantify interactions of 10 pairs of amides selected to complete this dataset. An analysis yields intrinsic strengths of six favorable and four unfavorable atom-atom interactions, expressed per unit area of each atom and relative to interactions with water. The most favorable interactions are sp2O-sp2C (lone pair-π, presumably n-π*), sp2C-sp2C (π-π and/or hydrophobic), sp2O-sp2N (hydrogen bonding) and sp3C-sp2C (CH-π and/or hydrophobic). Interactions of sp3C with itself (hydrophobic) and with sp2N are modestly favorable, while sp2N interactions with sp2N and with amide/aromatic sp2C are modestly unfavorable. Amide sp2O-sp2O interactions and sp2O-sp3C interactions are more unfavorable, indicating the preference of amide sp2O to interact with water. These intrinsic interaction strengths are used to predict interactions of amides with proteins and chemical effects of amides (including urea, N-ethylpyrrolidone [NEP], and polyvinylpyrrolidone [PVP]) on protein stability.

Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis.

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones.

Impact of pre-bypass ultrafiltration on prime values and clinical outcomes in neonatal and infant cardiopulmonary bypass.

BACKGROUND: A standard blood prime for cardiopulmonary bypass (CPB) in congenital cardiac surgery may possess non-physiologic values for electrolytes, glucose, and lactate. Pre-bypass Ultrafiltration (PBUF) can make these values more physiologic and standardized prior to bypass initiation. We aimed to determine if using PBUF on blood primes including packed red blood cells and thawed plasma would make prime values more predictable and physiologic. Additionally, we aimed to evaluate whether the addition of PBUF had an impact on outcome measures. METHODS: Retrospective review of consecutive patients ≤ 1 year of age undergoing an index cardiac operation on CPB between 8/2017 and 9/2021. As PBUF was performed at the perfusionists' discretion, a natural grouping of patients that received PBUF vs. those that did not occur. Differences in electrolytes, glucose, and lactate were compared at specific time points using Fisher's exact test for categorical variables and the Wilcoxon rank sum test for continuous variables. Clinical outcomes were also assessed. RESULTS: In both cohorts, the median age at surgery was 3 months and 47% of patients were female; 308/704 (44%) of the PBUF group and 163/414 (39%) of the standard prime group had at least one preoperative risk factor. The proportion of PBUF circuits which demonstrated more physiologic values for glucose (318 [45%]), sodium (434, [62%]), potassium (688 [98%]), lactate (612 [87%]) and osmolality (595 [92%]) was significantly higher when compared to standard prime circuit levels for glucose (8 [2%]), sodium (13 [3%], potassium (150 [36%]), lactate (56 [13%]) and osmolality (23 [6%]) prior to CPB initiation. There were no differences in clinical outcomes or rates of major adverse events between the two cohorts. CONCLUSIONS: PBUF creates standardized and more physiologic values for electrolytes, glucose, and lactate before the initiation of bypass without significant impacts on in-hospital outcomes.

Impaired B-cell tolerance checkpoints promote the development of autoimmune diseases and pathogenic autoantibodies.

A role for B cells in autoimmune diseases is now clearly established both in mouse models and humans by successful treatment of multiple sclerosis and rheumatoid arthritis with anti-CD20 monoclonal antibodies that eliminate B cells. However, the underlying mechanisms by which B cells promote the development of autoimmune diseases remain poorly understood. Here, we review evidence that patients with autoimmune disease suffer from defects in early B-cell tolerance checkpoints and therefore fail to counterselect developing autoreactive B cells. These B-cell tolerance defects are primary to autoimmune diseases and may result from altered B-cell receptor signaling and dysregulated T-cell/regulatory T-cell compartment. As a consequence, large numbers of autoreactive naive B cells accumulate in the blood of patients with autoimmune diseases and may promote autoimmunity through the presentation of self-antigen to T cells. In addition, new evidence suggests that this reservoir of autoreactive naive B cells contains clones that may develop into CD27- CD21-/lo B cells associated with increased disease severity and plasma cells secreting potentially pathogenic autoantibodies after the acquisition of somatic hypermutations that improve affinity for self-antigens.

An Emergent Population Code in Primary Auditory Cortex Supports Selective Attention to Spectral and Temporal Sound Features.

Textbook descriptions of primary sensory cortex (PSC) revolve around single neurons' representation of low-dimensional sensory features, such as visual object orientation in primary visual cortex (V1), location of somatic touch in primary somatosensory cortex (S1), and sound frequency in primary auditory cortex (A1). Typically, studies of PSC measure neurons' responses along few (one or two) stimulus and/or behavioral dimensions. However, real-world stimuli usually vary along many feature dimensions and behavioral demands change constantly. In order to illuminate how A1 supports flexible perception in rich acoustic environments, we recorded from A1 neurons while rhesus macaques (one male, one female) performed a feature-selective attention task. We presented sounds that varied along spectral and temporal feature dimensions (carrier bandwidth and temporal envelope, respectively). Within a block, subjects attended to one feature of the sound in a selective change detection task. We found that single neurons tend to be high-dimensional, in that they exhibit substantial mixed selectivity for both sound features, as well as task context. We found no overall enhancement of single-neuron coding of the attended feature, as attention could either diminish or enhance this coding. However, a population-level analysis reveals that ensembles of neurons exhibit enhanced encoding of attended sound features, and this population code tracks subjects' performance. Importantly, surrogate neural populations with intact single-neuron tuning but shuffled higher-order correlations among neurons fail to yield attention- related effects observed in the intact data. These results suggest that an emergent population code not measurable at the single-neuron level might constitute the functional unit of sensory representation in PSC.SIGNIFICANCE STATEMENT The ability to adapt to a dynamic sensory environment promotes a range of important natural behaviors. We recorded from single neurons in monkey primary auditory cortex (A1), while subjects attended to either the spectral or temporal features of complex sounds. Surprisingly, we found no average increase in responsiveness to, or encoding of, the attended feature across single neurons. However, when we pooled the activity of the sampled neurons via targeted dimensionality reduction (TDR), we found enhanced population-level representation of the attended feature and suppression of the distractor feature. This dissociation of the effects of attention at the level of single neurons versus the population highlights the synergistic nature of cortical sound encoding and enriches our understanding of sensory cortical function.

Kidney accelerated placement project: Outcomes and lessons learned.

Opportunities continue to be lost with a high rate of kidneys recovered for transplant but not utilized, particularly those considered less than ideal quality. The Organ Procurement and Transplantation Network (OPTN) Organ Center is tasked with allocating arguably the most difficult-to-place kidneys, and we hypothesized an accelerated placement pathway would increase utilization of kidneys placed by the Organ Center. The Kidney Accelerated Placement (KAP) project, implemented by the Organ Center from July 18, 2019 to July 15, 2020, aimed to offer kidneys with a high kidney donor profile index to programs that had a history of accepting such organs. We compared OPTN kidney match run, donor, and transplant recipient data during the project period and 1 year prior. There was no statistically significant change in the percentage of KAP-eligible donors accepted during the project period (16.4%) compared to the prior year (17.5%). Conversion from acceptance to transplant was higher under KAP (72.7% vs. 71.2%), though not significant. Waiting to accelerate placement after kidneys have been declined by multiple transplant programs locally and regionally is an intervention that may come too late to effectively increase utilization. Transplant rates of nationally shared and marginal kidneys remain a challenge, and future iterations of this project should be investigated.