Hypertonicity and/or acidosis induce marked rheological changes under hypoxic conditions in sickle trait red blood cells.

Deformability and sickling of red blood cells (RBCs) from individuals with sickle cell trait (SCT) was evaluated under harsh biophysical conditions that mimic certain vascular beds in vivo. RBC deformability in osmotic-gradient ektacytometry was decreased in HbAS (SCT) compared to HbAA (wild-type) RBCs at supraphysiological osmolalities. RBC deformability was also measured by oxygen-gradient ektacytometry. Whereas RBC sickling was not observed under isotonic and neutral pH conditions, hypertonicity and acidosis alone or in combination induced reversible sickling of SCT RBC. These data suggest that hyperosmolality and/or acidosis enhance hypoxia-induced sickling of SCT RBC.

Leaf cell wall properties and stomatal density influence oxygen isotope enrichment of leaf water.

Measurements of oxygen isotope enrichment of leaf water above source water (Δ18 OLW ) can improve our understanding of the interaction between leaf anatomy and physiology on leaf water transport. Models have been developed to predict Δ18 OLW such as the string-of-lakes model, which describes the mixing of leaf water pools, and the Péclet effect model, which incorporates transpiration rate and the mixing length between unenriched xylem and enriched mesophyll water in the mesophyll (Lm ) or veins (Lv ). Here we compare measurements and models of Δ18 OLW on two cell wall composition mutants grown under two light intensities and relative humidities to evaluate cell wall properties on leaf water transport. In maize (Zea mays), the compromised ultrastructure of the suberin lamellae in the bundle sheath of the ALIPHATIC SUBERIN FERULOYL TRANSFERASE mutant (Zmasft) reduced barriers to apoplastic water movement, resulting in higher E and, potentially, Lv and, consequently, lower Δ18 OLW . The difference in Δ18 OLW in cellulose synthase-like F6 (CslF6) mutants and wild-type of rice (Oryza sativa) grown under two light intensities co-varied with stomatal density. These results show that cell wall composition and stomatal density influence Δ18 OLW and that stable isotopes can facilitate the development of a physiologically and anatomically explicit water transport model.

Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways.

Storage lesion-induced, red cell-derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV-induced coagulation activation, the ability of storage lesion-induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV-induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Pan-cancer prediction of radiotherapy benefit using genomic-adjusted radiation dose (GARD): a cohort-based pooled analysis.

BACKGROUND: Despite advances in cancer genomics, radiotherapy is still prescribed on the basis of an empirical one-size-fits-all paradigm. Previously, we proposed a novel algorithm using the genomic-adjusted radiation dose (GARD) model to personalise prescription of radiation dose on the basis of the biological effect of a given physical dose of radiation, calculated using individual tumour genomics. We hypothesise that GARD will reveal interpatient heterogeneity associated with opportunities to improve outcomes compared with physical dose of radiotherapy alone. We aimed to test this hypothesis and investigate the GARD-based radiotherapy dosing paradigm. METHODS: We did a pooled, pan-cancer analysis of 11 previously published clinical cohorts of unique patients with seven different types of cancer, which are all available cohorts with the data required to calculate GARD, together with clinical outcome. The included cancers were breast cancer, head and neck cancer, non-small-cell lung cancer, pancreatic cancer, endometrial cancer, melanoma, and glioma. Our dataset comprised 1615 unique patients, of whom 1298 (982 with radiotherapy, 316 without radiotherapy) were assessed for time to first recurrence and 677 patients (424 with radiotherapy and 253 without radiotherapy) were assessed for overall survival. We analysed two clinical outcomes of interest: time to first recurrence and overall survival. We used Cox regression, stratified by cohort, to test the association between GARD and outcome with separate models using dose of radiation and sham-GARD (ie, patients treated without radiotherapy, but modelled as having a standard-of-care dose of radiotherapy) for comparison. We did interaction tests between GARD and treatment (with or without radiotherapy) using the Wald statistic. FINDINGS: Pooled analysis of all available data showed that GARD as a continuous variable is associated with time to first recurrence (hazard ratio [HR] 0·98 [95% CI 0·97-0·99]; p=0·0017) and overall survival (0·97 [0·95-0·99]; p=0·0007). The interaction test showed the effect of GARD on overall survival depends on whether or not that patient received radiotherapy (Wald statistic p=0·011). The interaction test for GARD and radiotherapy was not significant for time to first recurrence (Wald statistic p=0·22). The HR for physical dose of radiation was 0·99 (95% CI 0·97-1·01; p=0·53) for time to first recurrence and 1·00 (0·96-1·04; p=0·95) for overall survival. The HR for sham-GARD was 1·00 (0·97-1·03; p=1·00) for time to first recurrence and 1·00 (0·98-1·02; p=0·87) for overall survival. INTERPRETATION: The biological effect of radiotherapy, as quantified by GARD, is significantly associated with time to first recurrence and overall survival for patients with cancer treated with radiation. It is predictive of radiotherapy benefit, and physical dose of radiation is not. We propose integration of genomics into radiation dosing decisions, using a GARD-based framework, as the new paradigm for personalising radiotherapy prescription dose. FUNDING: None. VIDEO ABSTRACT.

A genetic link between leaf carbon isotope composition and whole-plant water use efficiency in the C4 grass Setaria.

Genetic selection for whole-plant water use efficiency (yield per transpiration; WUEplant ) in any crop-breeding programme requires high-throughput phenotyping of component traits of WUEplant such as intrinsic water use efficiency (WUEi ; CO2 assimilation rate per stomatal conductance). Measuring WUEi by gas exchange measurements is laborious and time consuming and may not reflect an integrated WUEi over the life of the leaf. Alternatively, leaf carbon stable isotope composition (δ13 Cleaf ) has been suggested as a potential time-integrated proxy for WUEi that may provide a tool to screen for WUEplant . However, a genetic link between δ13 Cleaf and WUEplant in a C4 species has not been well established. Therefore, to determine if there is a genetic relationship in a C4 plant between δ13 Cleaf and WUEplant under well watered and water-limited growth conditions, a high-throughput phenotyping facility was used to measure WUEplant in a recombinant inbred line (RIL) population created between the C4 grasses Setaria viridis and S. italica. Three quantitative trait loci (QTL) for δ13 Cleaf were found and co-localized with transpiration, biomass accumulation, and WUEplant . Additionally, WUEplant for each of the δ13 Cleaf QTL allele classes was negatively correlated with δ13 Cleaf , as would be predicted when WUEi influences WUEplant . These results demonstrate that δ13 Cleaf is genetically linked to WUEplant , likely to be through their relationship with WUEi , and can be used as a high-throughput proxy to screen for WUEplant in these C4 species.

Red blood cells modulate structure and dynamics of venous clot formation in sickle cell disease.

Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Erythrocyte sickling, the primary pathologic event in SCD, results in dramatic morphological changes in red blood cells (RBCs) because of polymerization of the abnormal hemoglobin. We used a mouse model of SCD and blood samples from sickle patients to determine if these changes affect the structure, properties, and dynamics of sickle clot formation. Sickling of RBCs and a significant increase in fibrin deposition were observed in venous thrombi formed in sickle mice. During ex vivo clot contraction, the number of RBCs extruded from sickle whole blood clots was significantly reduced compared with the number released from sickle cell trait and nonsickle clots in both mice and humans. Entrapment of sickled RBCs was largely factor XIIIa-independent and entirely mediated by the platelet-free cellular fraction of sickle blood. Inhibition of phosphatidylserine, but not administration of antisickling compounds, increased the number of RBCs released from sickle clots. Interestingly, whole blood, but not plasma clots from SCD patients, was more resistant to fibrinolysis, indicating that the cellular fraction of blood mediates resistance to tissue plasminogen activator. Sickle trait whole blood clots demonstrated an intermediate phenotype in response to tissue plasminogen activator. RBC exchange in SCD patients had a long-lasting effect on normalizing whole blood clot contraction. Furthermore, RBC exchange transiently reversed resistance of whole blood sickle clots to fibrinolysis, in part by decreasing platelet-derived PAI-1. These properties of sickle clots may explain the increased risk of venous thromboembolism observed in SCD.

Pregnancy in sickle cell trait: what we do and don't know.

Sickle cell trait (SCT) is the carrier state for sickle cell disease that results from the HBB rs334 missense mutation (p.Glu6Val) in the ß-globin chain of haemoglobin. While not associated with any impact on life expectancy, it has been established that SCT is associated with an increased risk of both venous thromboembolism (and in particular, pulmonary embolism) and chronic kidney disease. It is largely unknown what short- or long-term effect, if any, pregnancy has upon the risk or outcomes of these disorders. In addition, SCT has been linked with various adverse outcomes in pregnancy, ranging from maternal complications such as elevated risk of bacteriuria to potentially life-threatening entities such as pre-eclampsia and prematurity. In these scenarios also, no clear association with SCT has been established. Given the high worldwide prevalence of SCT, further studies addressing these issues are warranted.

Components of Water Use Efficiency Have Unique Genetic Signatures in the Model C4 Grass Setaria.

Plant growth and water use are interrelated processes influenced by genetically controlled morphological and biochemical characteristics. Improving plant water use efficiency (WUE) to sustain growth in different environments is an important breeding objective that can improve crop yields and enhance agricultural sustainability. However, genetic improvement of WUE using traditional methods has proven difficult due to the low throughput and environmental heterogeneity of field settings. To overcome these limitations, this study utilizes a high-throughput phenotyping platform to quantify plant size and water use of an interspecific Setaria italica × Setaria viridis recombinant inbred line population at daily intervals in both well-watered and water-limited conditions. Our findings indicate that measurements of plant size and water use are correlated strongly in this system; therefore, a linear modeling approach was used to partition this relationship into predicted values of plant size given water use and deviations from this relationship at the genotype level. The resulting traits describing plant size, water use, and WUE all were heritable and responsive to soil water availability, allowing for a genetic dissection of the components of plant WUE under different watering treatments. Linkage mapping identified major loci underlying two different pleiotropic components of WUE. This study indicates that alleles controlling WUE derived from both wild and domesticated accessions can be utilized to predictably modulate trait values given a specified precipitation regime in the model C4 genus Setaria.

Cell wall properties in Oryza sativa influence mesophyll CO2 conductance.

Diffusion of CO2 from the leaf intercellular air space to the site of carboxylation (gm ) is a potential trait for increasing net rates of CO2 assimilation (Anet ), photosynthetic efficiency, and crop productivity. Leaf anatomy plays a key role in this process; however, there are few investigations into how cell wall properties impact gm and Anet . Online carbon isotope discrimination was used to determine gm and Anet in Oryza sativa wild-type (WT) plants and mutants with disruptions in cell wall mixed-linkage glucan (MLG) production (CslF6 knockouts) under high- and low-light growth conditions. Cell wall thickness (Tcw ), surface area of chloroplast exposed to intercellular air spaces (Sc ), leaf dry mass per area (LMA), effective porosity, and other leaf anatomical traits were also analyzed. The gm of CslF6 mutants decreased by 83% relative to the WT, with c. 28% of the reduction in gm explained by Sc . Although Anet /LMA and Anet /Chl partially explained differences in Anet between genotypes, the change in cell wall properties influenced the diffusivity and availability of CO2 . The data presented here indicate that the loss of MLG in CslF6 plants had an impact on gm and demonstrate the importance of cell wall effective porosity and liquid path length on gm .

Relationship of leaf oxygen and carbon isotopic composition with transpiration efficiency in the C4 grasses Setaria viridis and Setaria italica.

Leaf carbon and oxygen isotope ratios can potentially provide a time-integrated proxy for stomatal conductance (gs) and transpiration rate (E), and can be used to estimate transpiration efficiency (TE). In this study, we found significant relationships of bulk leaf carbon isotopic signature (δ13CBL) and bulk leaf oxygen enrichment above source water (Δ18OBL) with gas exchange and TE in the model C4 grasses Setaria viridis and S. italica. Leaf δ13C had strong relationships with E, gs, water use, biomass, and TE. Additionally, the consistent difference in δ13CBL between well-watered and water-limited plants suggests that δ13CBL is effective in separating C4 plants with different availability of water. Alternatively, the use of Δ18OBL as a proxy for E and TE in S. viridis and S. italica was problematic. First, the oxygen isotopic composition of source water, used to calculate leaf water enrichment (Δ18OLW), was variable with time and differed across water treatments. Second, water limitations changed leaf size and masked the relationship of Δ18OLW and Δ18OBL with E. Therefore, the data collected here suggest that δ13CBL but not Δ18OBL may be an effective proxy for TE in C4 grasses.

Water uptake of Alaskan tundra evergreens during the winter-spring transition.

PREMISE OF THE STUDY: The cold season in the Arctic extends over 8 to 9 mo, yet little is known about vascular plant physiology during this period. Evergreen species photosynthesize under the snow, implying that they are exchanging water with the atmosphere. However, liquid water available for plant uptake may be limited at this time. The study objective was to determine whether evergreen plants are actively taking up water while under snow and/or immediately following snowmelt during spring thaw. METHODS: In two in situ experiments, one at the plot level and another at the individual species level, (2)H-labeled water was used as a tracer injected beneath the snow, after which plant stems and leaves were tested for the presence of the label. In separate experiments, excised shoots of evergreen species were exposed to (2)H-labeled water for ∼5 s or 60 min and tested for foliar uptake of the label. KEY RESULTS: In both the plot-level and the species-level experiments, some (2)H-labeled water was found in leaves and stems. Additionally, excised individual plant shoots exposed to labeled water for 60 min took up significantly more (2)H-label than shoots exposed ∼5 s. CONCLUSIONS: Evergreen tundra plants take up water under snow cover, some via roots, but also likely by foliar uptake. The ability to take up water in the subnivean environment allows evergreen tundra plants to take advantage of mild spring conditions under the snow and replenish carbon lost by winter respiration.

Emicizumab promotes factor Xa generation on endothelial cells.

BACKGROUND: Until recently, the treatment of hemophilia A relied on factor (F)VIII replacement. However, up to one-third of patients with severe hemophilia A develop neutralizing alloantibodies that render replacement therapies ineffective. The development of emicizumab, a bispecific antibody that partially mimics FVIIIa, has revolutionized the treatment of these patients. However, the use of an activated prothrombin complex concentrate [FEIBA (Takeda)] to treat breakthrough bleeding in patients on emicizumab has been associated with thrombotic complications including a unique microangiopathy. OBJECTIVES: We hypothesized that the thrombotic complications observed with the combination of emicizumab and FEIBA might be due to excessive expression of procoagulant activity on the surface of endothelial cells. METHODS: We examined the ability of emicizumab to promote FX activation on endothelial cells using 2 cell culture models. RESULTS: We found that endothelial cells readily support emicizumab-mediated activation of FX by FIXa. The level of FXa generation depends on the concentration of available FIXa. The addition of FEIBA to emicizumab increased FXa generation in a dose-dependent manner on endothelial cells in both models. The rate of FXa generation was further enhanced by endothelial cell activation. However, unlike emicizumab, we found limited FXa generation in the presence of FVIII(a), which followed a significant lag time and was not dependent on FIXa concentration under these conditions. CONCLUSION: Emicizumab promotes FXa generation on the surface of endothelial cells, which is markedly enhanced in the presence of FEIBA. These findings demonstrate a potential mechanism for the thrombotic complications seen with the combined use of emicizumab and FEIBA.

The role of effective leaf mixing length in the relationship between the δ18 O of stem cellulose and source water across a salinity gradient.

Previous mangrove tree ring studies attempted, unsuccessfully, to relate the δ(18) O of trunk cellulose (δ(18) O(CELL) ) to the δ(18) O of source water (δ(18) O(SW) ). Here, we tested whether biochemical fractionation associated with one of the oxygen in the cellulose glucose moiety or variation in leaf water oxygen isotope fractionation (Δ(LW) ) can interfere with the δ(18) O(SW) signal as it is recorded in the δ(18) O(CELL) of mangrove (saltwater) and hammock (freshwater) plants. We selected two transects experiencing a salinity gradient, located in the Florida Keys, USA. The δ(18) O(CELL) throughout both transects did not show the pattern expected based on that of the δ(18) O(SW) . We found that in one of the transects, biochemical fractionation interfered with the δ(18) O(SW) signal, while in the other transect Δ(LW) differed between mangrove and hammock plants. Observed differences in Δ(LW) between mangroves and hammocks were caused by a longer effective leaf mixing length (L) of the water pathway in mangrove leaves compared to those of hammock leaves. Changes in L could have caused the δ(18) O(CELL) to record not only variations in the δ(18) O(SW) but also in Δ(LW) making it impossible to isolate the δ(18) O(SW) signal.

Indicators of water use efficiency across diverse agroecosystems and spatiotemporal scales.

Understanding the relationship between water and production within and across agroecosystems is essential for addressing several agricultural challenges of the 21st century: providing food, fuel, and fiber to a growing human population, reducing the environmental impacts of agricultural production, and adapting food systems to climate change. Of all human activities, agriculture has the highest demand for water globally. Therefore, increasing water use efficiency (WUE), or producing 'more crop per drop', has been a long-term goal of agricultural management, engineering, and crop breeding. WUE is a widely used term applied across a diverse array of spatial scales, spanning from the leaf to the globe, and over temporal scales ranging from seconds to months to years. The measurement, interpretation, and complexity of WUE varies enormously across these spatial and temporal scales, challenging comparisons within and across diverse agroecosystems. The goals of this review are to evaluate common indicators of WUE in agricultural production and assess tradeoffs when applying these indicators within and across agroecosystems amidst a changing climate. We examine three questions: (1) what are the uses and limitations of common WUE indicators, (2) how can WUE indicators be applied within and across agroecosystems, and (3) how can WUE indicators help adapt agriculture to climate change? Addressing these agricultural challenges will require land managers, producers, policy makers, researchers, and consumers to evaluate costs and benefits of practices and innovations of water use in agricultural production. Clearly defining and interpreting WUE in the most scale-appropriate way is crucial for advancing agroecosystem sustainability.

Measuring competitive exclusion in non-small cell lung cancer.

In this study, we experimentally measure the frequency-dependent interactions between a gefitinib-resistant non-small cell lung cancer population and its sensitive ancestor via the evolutionary game assay. We show that cost of resistance is insufficient to accurately predict competitive exclusion and that frequency-dependent growth rate measurements are required. Using frequency-dependent growth rate data, we then show that gefitinib treatment results in competitive exclusion of the ancestor, while the absence of treatment results in a likely, but not guaranteed, exclusion of the resistant strain. Then, using simulations, we demonstrate that incorporating ecological growth effects can influence the predicted extinction time. In addition, we show that higher drug concentrations may not lead to the optimal reduction in tumor burden. Together, these results highlight the potential importance of frequency-dependent growth rate data for understanding competing populations, both in the laboratory and as we translate adaptive therapy regimens to the clinic.

Factor-mimetic and rebalancing therapies in hemophilia A and B: the end of factor concentrates?

Hemophilia A (HA) and B are inherited bleeding disorders caused by a deficiency of factor VIII or factor IX, respectively. The current standard of care is the administration of recombinant or purified factor. However, this treatment strategy still results in a high economic and personal burden to patients, which is further exacerbated by the development of inhibitors-alloantibodies to factor. The treatment landscape is changing, with nonfactor therapeutics playing an increasing role in what we consider to be the standard of care. Emicizumab, a bispecific antibody that mimics the function of factor VIIIa, is the first such nonfactor therapy to gain US Food and Drug Administration approval and is rapidly changing the paradigm for HA treatment. Other therapies on the horizon seek to target anticoagulant proteins in the coagulation cascade, thus "rebalancing" a hemorrhagic tendency by introducing a thrombotic tendency. This intricate hemostatic balancing act promises great things for patients in need of more treatment options, but are these other therapies going to replace factor therapy? In light of the many challenges facing these therapies, should they be viewed as a replacement of our current standard of care? This review discusses the background, rationale, and potential of nonfactor therapies as well as the anticipated pitfalls and limitations. This is done in the context of a review of our current understanding of the many aspects of the coagulation system.

Recombinant porcine FVIII for bleed treatment in acquired hemophilia A: findings from a single-center, 18-patient cohort.

Acquired hemophilia A (AHA) is a rare bleeding disorder in which acquired autoantibodies to endogenous factor VIII (FVIII) decrease FVIII activity and lead to a bleeding phenotype. A substantial majority of individuals who develop AHA present with severe bleeding. Effective treatment requires both immunosuppressive therapy and prompt hemostatic treatment. Bleeding is commonly treated with bypassing agents (BPAs) such as recombinant activated FVII (rFVIIa) or activated prothrombin complex concentrates Disadvantages to BPAs include the inability to monitor response with standard laboratory assays, inconsistent hemostatic efficacy, and thrombosis. Recombinant porcine FVIII (rpFVIII: Obizur, Baxter, Deerfield, IL) was approved by the US Food and Drug Administration (FDA) for bleed treatment in AHA in 2014, and has the advantage of laboratory monitoring of FVIII activity levels and known hemostatic efficacy in the presence of anti-human FVIII inhibitors and after failure of BPAs. Using an algorithm-based approach, rpFVIII has been used to successfully treat 18 patients with AHA at our center with substantially lower doses than the current FDA-recommended dosing. Additionally, data from our cohort show that the preexposure anti-porcine Bethesda titer does not reliably predict the clinical response to rpFVIII treatment and is not correlated with the anti-human Bethesda titer. We also present data showing lower total rpFVIII use for initial bleed resolution when rpVIII is used upfront, as compared with use as rescue therapy. We validated our dosing algorithm, which uses much lower than FDA-recommended doses with 14 more patients than in our previously reported patient series.