Hypoxia drives CD39-dependent suppressor function in exhausted T cells to limit antitumor immunity.

CD8+ T cells are critical for elimination of cancer cells. Factors within the tumor microenvironment (TME) can drive these cells to a hypofunctional state known as exhaustion. The most terminally exhausted T (tTex) cells are resistant to checkpoint blockade immunotherapy and might instead limit immunotherapeutic efficacy. Here we show that intratumoral CD8+ tTex cells possess transcriptional features of CD4+Foxp3+ regulatory T cells and are similarly capable of directly suppressing T cell proliferation ex vivo. tTex cell suppression requires CD39, which generates immunosuppressive adenosine. Restricted deletion of CD39 in endogenous CD8+ T cells resulted in slowed tumor progression, improved immunotherapy responsiveness and enhanced infiltration of transferred tumor-specific T cells. CD39 is induced on tTex cells by tumor hypoxia, thus mitigation of hypoxia limits tTex suppression. Together, these data suggest tTex cells are an important regulatory population in cancer and strategies to limit their generation, reprogram their immunosuppressive state or remove them from the TME might potentiate immunotherapy.

Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion.

Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.

Regulation and Immunotherapeutic Targeting of the Epigenome in Exhausted CD8 T Cell Responses.

Exhaustion is a state of CD8 T cell differentiation that occurs in settings of chronic Ag such as tumors, chronic viral infection, and autoimmunity. Cellular differentiation is driven by a series of environmental signals that promote epigenetic landscapes that set transcriptomes needed for function. For CD8 T cells, the epigenome that underlies exhaustion is distinct from effector and memory cell differentiation, suggesting that signals early on set in motion a process where the epigenome is modified to promote a trajectory toward a dysfunctional state. Although we know many signals that promote exhaustion, putting this in the context of the epigenetic changes that occur during differentiation has been less clear. In this review, we aim to summarize the epigenetic changes associated with exhaustion in the context of signals that promote it, highlighting immunotherapeutic studies that support these observations or areas for future therapeutic opportunities.

Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells.

The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.

A framework for mapping and monitoring human-ocean interactions in near real-time during COVID-19 and beyond.

The human response to the COVID-19 pandemic set in motion an unprecedented shift in human activity with unknown long-term effects. The impacts in marine systems are expected to be highly dynamic at local and global scales. However, in comparison to terrestrial ecosystems, we are not well-prepared to document these changes in marine and coastal environments. The problems are two-fold: 1) manual and siloed data collection and processing, and 2) reliance on marine professionals for observation and analysis. These problems are relevant beyond the pandemic and are a barrier to understanding rapidly evolving blue economies, the impacts of climate change, and the many other changes our modern-day oceans are undergoing. The "Our Ocean in COVID-19″ project, which aims to track human-ocean interactions throughout the pandemic, uses the new eOceans platform (eOceans.app) to overcome these barriers. Working at local scales, a global network of ocean scientists and citizen scientists are collaborating to monitor the ocean in near real-time. The purpose of this paper is to bring this project to the attention of the marine conservation community, researchers, and the public wanting to track changes in their area. As our team continues to grow, this project will provide important baselines and temporal patterns for ocean conservation, policy, and innovation as society transitions towards a new normal. It may also provide a proof-of-concept for real-time, collaborative ocean monitoring that breaks down silos between academia, government, and at-sea stakeholders to create a stronger and more democratic blue economy with communities more resilient to ocean and global change.

Alpha galactosidase A activity in Parkinson's disease.

Glucocerebrosidase (GCase, deficient in Gaucher disease) enzymatic activity measured in dried blood spots of Parkinson's Disease (PD) cases is within healthy range but reduced compared to controls. It is not known whether activities of additional lysosomal enzymes are reduced in dried blood spots in PD. To test whether reduction in lysosomal enzymatic activity in PD is specific to GCase, we measured GCase, acid sphingomyelinase (deficient in Niemann-Pick disease types A and B), alpha galactosidase A (deficient in Fabry), acid alpha-glucosidase (deficient in Pompe) and galactosylceramidase (deficient in Krabbe) enzymatic activities in dried blood spots of PD patients (n = 648) and controls (n = 317) recruited from Columbia University. Full sequencing of glucocerebrosidase (GBA) and the LRRK2 G2019S mutation was performed. Enzymatic activities were compared between PD cases and controls using t-test and regression models adjusted for age, gender, and GBA and LRRK2 G2019S mutation status. Alpha galactosidase A activity was lower in PD cases compared to controls both when only non-carriers were included (excluding all GBA and LRRK2 G2019S carriers and PD cases with age-at-onset below 40) [2.85 µmol/l/h versus 3.12 µmol/l/h, p = 0.018; after controlling for batch effect, p = 0.006 (468 PD cases and 296 controls)], and when including the entire cohort (2.89 µmol/l/h versus 3.10 µmol/l/h, p = 0.040; after controlling for batch effect, p = 0.011). Because the alpha galactosidase A gene is X-linked, we stratified the analyses by sex. Among women who were non-carriers of GBA and LRRK2 G2019S mutations (PD, n = 155; control, n = 194), alpha galactosidase A activity was lower in PD compared to controls (2.77 µmol/l/h versus 3.10 µmol/l/h, p = 0.044; after controlling for a batch effect, p = 0.001). The enzymatic activity of acid sphingomyelinase, acid alpha-glucosidase and galactosylceramidase was not significantly different between PD and controls. In non-carriers, most lysosomal enzyme activities were correlated, with the strongest association in GCase, acid alpha-glucosidase, and alpha galactosidase A (Pearson correlation coefficient between 0.382 and 0.532). In a regression model with all five enzymes among non-carriers (adjusted for sex and age), higher alpha galactosidase A activity was associated with lower odds of PD status (OR = 0.54; 95% CI:0.31-0.95; p = 0.032). When LRRK2 G2019S PD carriers (n = 37) were compared to non-carriers with PD, carriers had higher GCase, acid sphingomyelinase and alpha galactosidase A activity. We conclude that alpha galactosidase A may have a potential independent role in PD, in addition to GCase.

Quality improvement in hospitals: barriers and facilitators.

Purpose The purpose of this paper is to examine quality improvement (QI) initiatives in acute care hospitals, the factors associated with success, and the impacts on patient care and safety. Design/methodology/approach An extensive online survey was completed by senior managers responsible for QI. The survey assessed QI project types, QI methods, staff engagement, and barriers and factors in the success of QI initiatives. Findings The response rate was 37 percent, 46 surveys were completed from 125 acute care hospitals. QI initiatives had positive impacts on patient safety and care. Staff in all hospitals reported conducting past or present hand-hygiene QI projects and C. difficile and surgical site infection were the next most frequent foci. Hospital staff not having time and problems with staff prioritizing QI with other duties were identified as important QI barriers. All respondents reported hospital leadership support, data utilization and internal champions as important QI facilitators. Multiple regression models identified nurses' active involvement and medical staff engagement in QI with improved patient care and physicians' active involvement and medical staff engagement with greater patient safety. Practical implications There is the need to study how best to support and encourage physicians and nurses to become more engaged in QI. Originality/value QI initiatives were shown to have positive impacts on patient safety and patient care and barriers and facilitating factors were identified. The results indicated patient care and safety would benefit from increased physician and nurse engagement in QI initiatives.

Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively.

Evaluating the effectiveness of teeth and dorsal fin spines for non-lethal age estimation of a tropical reef fish, coral trout Plectropomus leopardus.

This study investigated whether teeth and dorsal fin spines could be used as non-lethal methods of age estimation for a vulnerable and highly valued tropical fisheries species, coral trout Plectropomus leopardus. Age estimation of individuals from 2 to 9 years old revealed that dorsal spines represent an accurate ageing method (90% agreement with otoliths) that was more precise [average per cent error (APE) = 4·1, coefficient of variation (c.v.) = 5·8%] than otoliths (APE = 6·2, c.v. = 8·7%). Of the three methods for age estimation (otoliths, dorsal spines and teeth), spines were the most time and cost efficient. An aquarium-based study also found that removing a dorsal spine or tooth did not affect survivorship or growth of P. leopardus. No annuli were visible in teeth despite taking transverse and longitudinal sections throughout the tooth and trialling several different laboratory methods. Although teeth may not be suitable for estimating age of P. leopardus, dorsal spines appear to be an acceptably accurate, precise and efficient method for non-lethal ageing of individuals from 2 to 9 years old in this tropical species.

Exploring light-emitting diode pumped luminescent concentrators in solid-state laser applications.

In the past, there were limited efforts to use light-emitting diodes (LEDs) for pumping solid-state lasers. However, these attempts were overshadowed by the introduction of laser diodes, which offered more favourable pumping conditions. Nevertheless, recent advancements in high-power LEDs, coupled with the utilization of luminescent concentrators (LC), have paved the way for a novel approach to pump solid-state lasers. The combination of LEDs and LC in this LED-LC system presents several advantages, including enhanced ruggedness, stability, and cost-effectiveness compared to other laser pumping methods. This review explores the various techniques employed to pump solid-state lasers using LED-LC as a pump source, along with improvements made to enhance the brightness of LEDs in this context.

Altered functional B cell subset populations in patients with chronic fatigue syndrome compared to healthy controls.

Chronic fatigue syndrome (CFS) is a heterogeneous disorder of unknown aetiology characterized by disabling fatigue, headaches, sleep disturbance and several other symptoms. The onset of CFS may follow a viral infection or period of stress. Patients with CFS do not have hypogammaglobulinaemia, predisposition to recurrent bacterial infections or symptoms of autoimmunity. To date, defects in B cell numbers or function have not been shown in the literature. However, treatment with anti-B cell therapy using Rituximab has recently shown benefit to CFS patients. We therefore postulated that patients with CFS had a subtle humoral immune dysfunction, and performed extended B cell immunophenotyping. We undertook a detailed characterization of the proportions of the different B cell subsets in 33 patients with CFS fulfilling the Canadian and Fukada criteria for CFS and compared these with 24 age- and gender-matched healthy controls (HC). CFS patients had greater numbers of naive B cells as a percentage of lymphocytes: 6·3 versus 3·9% in HC (P = 0·034), greater numbers of naive B cells as a percentage of B cells: 65 versus 47% in controls (P = 0·003), greater numbers of transitional B cells: 1·8 versus 0·8% in controls (P = 0·025) and reduced numbers of plasmablasts: 0·5 versus 0·9% in controls (P = 0·013). While the cause of these changes is unclear, we speculate whether they may suggest a subtle tendency to autoimmunity.

Manipulating avatar age and gender in level-2 visual perspective taking.

Visual perspective taking (VPT) represents how the world appears from another person's position. The age, group status and emotional displays of the other person have been shown to affect task performance, but tasks often confound social and spatial outcome measures by embedding perspective taking in explicitly social contexts or theory-of-mind reasoning. Furthermore, while previous research has suggested that visual perspective taking may be impacted by avatar characteristics, it is unknown whether this is driven by general group processing or a specific deficit in mentalizing about outgroups, for example, children. Therefore, using a minimally social task (i.e., the task was not communicative, and acknowledging the "mind" of the avatar was not necessitated), we examined whether avatar age and avatar gender affect performance on simpler (low angular disparity) and more effortful, embodied (high angular disparity) perspective judgments. Ninety-two participants represented the visuospatial perspectives of a boy, girl, man, or woman who were presented at various angular disparities. A target object was placed in front of the avatar and participants responded to the orientation of the object from the avatar's position. The findings suggest that social features of visuospatial perspective taking (VSPT) are processed separately from the fundamental spatial computations. Further, Level-2 VSPT appears to be affected by general group categorization (e.g., age and gender) rather than a deficit in mentalizing about a specific outgroup (e.g., children).

Chronic fatigue syndrome, the immune system and viral infection.

The chronic fatigue syndrome (CFS), as defined by recent criteria, is a heterogeneous disorder with a common set of symptoms that often either follows a viral infection or a period of stress. Despite many years of intense investigation there is little consensus on the presence, nature and degree of immune dysfunction in this condition. However, slightly increased parameters of inflammation and pro-inflammatory cytokines such as interleukin (IL) 1, IL6 and tumour necrosis factor (TNF) α are likely present. Additionally, impaired natural killer cell function appears evident. Alterations in T cell numbers have been described by some and not others. While the prevalence of positive serology for the common herpes viruses appears no different from healthy controls, there is some evidence of viral persistence and inadequate containment of viral replication. The ability of certain herpes viruses to impair the development of T cell memory may explain this viral persistence and the continuation of symptoms. New therapies based on this understanding are more likely to produce benefit than current methods.

Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory.

The identification and/or prediction of the antimicrobial resistance of microorganisms in clinical materials solely by molecular means in the diagnostic microbiology laboratory is not novel. However, the ability to sequence multitudes of bacterial genomes and deliver and interpret the resultant sequence information in near "real-time" is the basis of next-generation sequencing (NGS) technologies. There have been numerous applications and successes of NGS applications in the clinical and public health domain. However, none have, as yet, delivered perhaps the most sought after application, i.e., the generation of microbial sequence data for "real-time" patient management. In this review, we discuss the use of NGS and whole-genome sequencing (WGS) of microorganisms as a logical next step for the routine diagnosis of infection and the prediction of antimicrobial susceptibility in the clinical microbiology laboratory.

Tumor microenvironmental signals reshape chromatin landscapes to limit the functional potential of exhausted T cells.

Response rates to immunotherapy in solid tumors remain low due in part to the elevated prevalence of terminally exhausted T cells, a hypofunctional differentiation state induced through persistent antigen and stress signaling. However, the mechanisms promoting progression to terminal exhaustion in the tumor remain undefined. Using the low-input chromatin immunoprecipitation sequencing method CUT&RUN, we profiled the histone modification landscape of tumor-infiltrating CD8+ T cells throughout differentiation. We found that terminally exhausted T cells had unexpected chromatin features that limit their transcriptional potential. Terminally exhausted T cells had a substantial fraction of active chromatin, including active enhancers enriched for bZIP/AP-1 transcription factor motifs that lacked correlated gene expression, which was restored by immunotherapeutic costimulatory signaling. Reduced transcriptional potential was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Enforced expression of the hypoxia-insensitive histone demethylase Kdm6b was sufficient to overcome hypoxia, increase function, and promote antitumor immunity. Our study reveals the specific epigenetic changes mediated by histone modifications during T cell differentiation that support exhaustion in cancer, highlighting that their altered function is driven by improper costimulatory signals and environmental factors. These data suggest that even terminally exhausted T cells may remain competent for transcription in settings of increased costimulatory signaling and reduced hypoxia.

Evidence-based approach for interpretation of Epstein-Barr virus serological patterns.

Diagnosis of Epstein-Barr virus (EBV) infection is based on clinical symptoms and serological markers, including the following: immunoglobulin G (IgG) and IgM antibodies to the viral capsid antigen (VCA), heterophile antibodies, and IgG antibodies to the EBV early antigen-diffuse (EA-D) and nuclear antigen (EBNA-1). The use of all five markers results in 32 possible serological patterns. As a result, interpretation of EBV serologies remains a challenge. The purpose of this study was to use a large population of patients to develop evidence-based tools for interpreting EBV results. This study utilized 1,846 serum specimens sent to the laboratory for physician-ordered EBV testing. Chart review was performed for more than 800 patients, and diagnoses were assigned based on physician-ordered testing, clinical presentation, and patient history. Testing for all five EBV antibodies was performed separately on all serum samples using the Bio-Rad BioPlex 2200 system. Presumed EBV diagnosis (based on previous publications) was compared to EBV diagnosis based on a medical record review for each serological pattern. Interestingly, of the 32 possible serological patterns, only 12 occurred in > or = 10 patients. The remaining 20 patterns were uninterpretable because they occurred with such infrequency. Two easy-to-use tables were created to interpret EBV serological patterns based on whether three (EBV VCA IgG, IgM, and heterophile) or five markers are utilized. The use of these two tables allows for interpretation of >95% of BioPlex serological results. This is the first evidence-based study of its kind for EBV serology.

Breeding cycle of the flea Cediopsylla simplex is controlled by breeding cycle of host.

Maturation of female Cediopsylla simplex takes place on the pregnant rabbit and nestlings but not on the estrous doe. If matured fleas are transferred to the estrous doe their ovaries are resorbed.

Pollination and fruit set in two rewardless slipper orchids and their hybrids (Cypripedium, Orchidaceae): large yellow flowers outperform small white flowers in the northern tall grass prairie.

Species with rewardless flowers often have low fruit to flower ratios, although wide temporal and spatial variation in fruiting success can occur. We compared floral phenotypes, insect visitors and fruiting success in four populations of the small white (Cypripedium candidum) and yellow (C. parviflorum) lady's slipper orchids and their hybrids near the northern extent of North America's tall grass prairie. Flower and fruit numbers were observed for two seasons on marked individuals (n = 1811). Floral traits were measured on 82-140 individuals per taxon and analysed in relation to fruiting success. All insects found inside flowers were collected, inspected for pollen smears and measured for comparison to floral features. Among orchid taxa, C. candidum had the smallest flowers, lowest number and variety of insect visitors, and lowest fruit to flower ratios. These measures were intermediate in hybrids and highest in C. parviflorum, despite low flower numbers in the latter. Within orchid taxa, fruit number was positively related to flower number, but fruit to flower ratios decreased slightly, as would be expected if pollinators left unrewarding patches. Potential pollinators included the dipteran Odontomyia pubescens and hymenopterans Andrena spp., Apis mellifera and Lasioglossum zonulum. Cypripedium parviflorum had a reproductive advantage over C. candidum across multiple populations and years. Hybrids showed segregation for floral traits, and hybrid fruiting success increased with a deeper intensity of yellow pigment and larger escape routes for floral visitors. These same attributes likely contributed to the relatively high fruit set in C. parviflorum in the study region.

Elevated PTEN expression maintains anergy in human B cells and reveals unexpectedly high repertoire autoreactivity.

It has been reported that 2.5%-30% of human peripheral CD27- B cells are autoreactive and anergic based on unresponsiveness to antigen receptor (BCR) stimulation and autoreactivity of cloned and expressed BCR. The molecular mechanisms that maintain this unresponsiveness are unknown. Here, we showed that in humans anergy is maintained by elevated expression of PTEN, a phosphatidylinositol 3,4,5P-3-phosphatase. Upregulation of PTEN was associated with reduced expression of microRNAs that control its expression. Pharmacologic inhibition of PTEN lead to significant restoration of responsiveness. Consistent with a role in conferring risk of autoimmunity, B cells from type 1 diabetics and autoimmune thyroid disease patients expressed reduced PTEN. Unexpectedly, in healthy individuals PTEN expression was elevated in on average 40% of CD27- B cells, with levels gradually decreasing as IgM levels increase. Our findings suggest that a much higher proportion of the peripheral repertoire is autoreactive than previously thought and that B cells upregulate PTEN in a manner that is proportional to the recognition of autoantigens of increasing avidity, thus tuning BCR signaling to prevent development of autoimmunity while providing a reservoir of cells that can be readily activated to respond when needed.

Future Fire Impacts on Smoke Concentrations, Visibility, and Health in the Contiguous United States.

Fine particulate matter (PM2.5) from U.S. anthropogenic sources is decreasing. However, previous studies have predicted that PM2.5 emissions from wildfires will increase in the midcentury to next century, potentially offsetting improvements gained by continued reductions in anthropogenic emissions. Therefore, some regions could experience worse air quality, degraded visibility, and increases in population-level exposure. We use global climate model simulations to estimate the impacts of changing fire emissions on air quality, visibility, and premature deaths in the middle and late 21st century. We find that PM2.5 concentrations will decrease overall in the contiguous United States (CONUS) due to decreasing anthropogenic emissions (total PM2.5 decreases by 3% in Representative Concentration Pathway [RCP] 8.5 and 34% in RCP4.5 by 2100), but increasing fire-related PM2.5 (fire-related PM2.5 increases by 55% in RCP4.5 and 190% in RCP8.5 by 2100) offsets these benefits and causes increases in total PM2.5 in some regions. We predict that the average visibility will improve across the CONUS, but fire-related PM2.5 will reduce visibility on the worst days in western and southeastern U.S. regions. We estimate that the number of deaths attributable to total PM2.5 will decrease in both the RCP4.5 and RCP8.5 scenarios (from 6% to 4-5%), but the absolute number of premature deaths attributable to fire-related PM2.5 will double compared to early 21st century. We provide the first estimates of future smoke health and visibility impacts using a prognostic land-fire model. Our results suggest the importance of using realistic fire emissions in future air quality projections.