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Heat-flow mapping of the western USA has identified an apparent low-heat-flow anomaly coincident with the Columbia Plateau Regional Aquifer System, a thick sequence of basalt aquifers within the Columbia River Basalt Group ( CRBG). A heat and mass transport model ( SUTRA) was used to evaluate the potential impact of groundwater flow on heat flow along two different regional groundwater flow paths. Limited in situ permeability ( k) data from the CRBG are compatible with a steep permeability decrease (approximately 3.5 orders of magnitude) at 600-900 m depth and approximately 40°C. Numerical simulations incorporating this permeability decrease demonstrate that regional groundwater flow can explain lower-than-expected heat flow in these highly anisotropic ( kx/ kz ~ 104) continental flood basalts. Simulation results indicate that the abrupt reduction in permeability at approximately 600 m depth results in an equivalently abrupt transition from a shallow region where heat flow is affected by groundwater flow to a deeper region of conduction-dominated heat flow. Most existing heat-flow measurements within the CRBG are from shallower than 600 m depth or near regional groundwater discharge zones, so that heat-flow maps generated using these data are likely influenced by groundwater flow. Substantial k decreases at similar temperatures have also been observed in the volcanic rocks of the adjacent Cascade Range volcanic arc and at Kilauea Volcano, Hawaii, where they result from low-temperature hydrothermal alteration. [ABSTRACT FROM AUTHOR]

Background: Apixaban is commonly used to prevent stroke in older adults with nonvalvular atrial fibrillation (AF). Although its package insert has specific dose reduction criteria, providers may dose reduce outside of these parameters based on clinical scenarios., Objective: The primary objective was to determine the incidence of apixaban off-label reduced dosing, while secondarily determining the safety and efficacy outcomes associated with such dosing., Methods: A retrospective analysis of patients aged 65 and older with orders for apixaban for AF was institutional review board (IRB)-approved and conducted across 3 academic medical centers. Patients receiving off-label reduced-dose apixaban (ie, "underdosed") were matched to a cohort of patients dosed according to the package insert at the standard dosing (5 mg twice daily) using stratified random sampling. Secondary outcomes included 1-year incidence of major bleeding, clinically relevant non-major bleeding (CRNMB), stroke or transient ischemic attack (TIA), and mortality. The Fisher exact tests were used to compare between-group differences., Results: Of the 1172 patients meeting initial inclusion criteria, 201 (17%) were dosed off-label, with 175 (15%) "underdosed." The 147 "underdosed" patients with documented follow-up were matched with 139 patients receiving standard Food and Drug Administration (FDA)-labeled dosing. There were no significant differences in incidence of stroke (2.7% vs 2.2%), major bleeding (0% vs 0.7%), and CRNMB (2.7% vs 1.4%) in the off-label reduced dosing versus standard dosing groups. All-cause mortality was higher in the off-label reduced-dose group (16 [10.9%] vs 2 [1.4%], P < 0.05)., Conclusion and Relevance: Older adults with nonvalvular AF are commonly prescribed lower-than-recommended doses of apixaban. However, no significant association was found between empiric off-label reduced dosing and stroke or bleeding outcomes., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To achieve reductions in infusion time, infusion sites, and frequency, a prospective, open-label, multicenter, Phase 3 study evaluated the safety, efficacy, and tolerability of subcutaneous immunoglobulin (SCIG) 16.5% (Cutaquig®, Octapharma) at enhanced infusion regimens., Methods: Three separate cohorts received SCIG 16.5% evaluating volume, rate, and frequency: Cohort 1) volume assessment/site: up to a maximum 100 mL/site; Cohort 2) infusion flow rate/site: up to a maximum of 100 mL/hr/site or the maximum flow rate achievable by the tubing; Cohort 3) infusion frequency: every other week at twice the patient's weekly dose., Results: For Cohort 1 (n = 15), the maximum realized volume per site was 108 mL/site, exceeding the currently labeled (US) maximum (up to 40 mL/site for adults). In Cohort 2 (n = 15), the maximum realized infusion flow rate was 67.5 mL/hr/site which is also higher than the labeled (US) maximum (up to 52 mL/hr/site). In Cohort 3 (n = 34), the mean total trough levels for every other week dosing demonstrated equivalency to weekly dosing (p value = 0.0017). All regimens were well tolerated. There were no serious bacterial infections (SBIs). Most patients had mild (23.4%) or moderate (56.3%) adverse events. The majority of patients found the new infusion regimens to be better or somewhat better than their previous regimens and reported that switching to SCIG 16.5% was easy., Conclusions: SCIG 16.5% (Cutaquig®), infusions are efficacious, safe, and well tolerated with reduced infusion time, fewer infusion sites, and reduced frequency. Further, the majority of patients found the new infusion regimens to be better or somewhat better than their previous regimens., (© 2023. The Author(s).)

Net-zero greenhouse gas (GHG) emissions targets are driving interest in opportunities for biomass-based negative emissions and bioenergy, including from marine sources such as seaweed. Yet the biophysical and economic limits to farming seaweed at scales relevant to the global carbon budget have not been assessed in detail. We use coupled seaweed growth and technoeconomic models to estimate the costs of global seaweed production and related climate benefits, systematically testing the relative importance of model parameters. Under our most optimistic assumptions, sinking farmed seaweed to the deep sea to sequester a gigaton of CO 2 per year costs as little as US$480 per tCO 2 on average, while using farmed seaweed for products that avoid a gigaton of CO 2 -equivalent GHG emissions annually could return a profit of $50 per tCO 2 -eq. However, these costs depend on low farming costs, high seaweed yields, and assumptions that almost all carbon in seaweed is removed from the atmosphere (that is, competition between phytoplankton and seaweed is negligible) and that seaweed products can displace products with substantial embodied non-CO 2 GHG emissions. Moreover, the gigaton-scale climate benefits we model would require farming very large areas (>90,000 km 2 )-a >30-fold increase in the area currently farmed. Our results therefore suggest that seaweed-based climate benefits may be feasible, but targeted research and demonstrations are needed to further reduce economic and biophysical uncertainties., (© 2022. The Author(s).)

Policies requiring childcare settings to promote healthy eating, physical activity, and limited screentime have the potential to improve young children's health. However, policies may have limited impact without effective implementation strategies to promote policy adoption. In this mixed-methods study, we evaluated the type, quality, and dose of implementation strategies for state-level childcare licensing regulations focused on healthy eating, physical activity, or screentime using: (1) a survey of state licensing staff and technical assistance providers (n = 89) in 32 states; (2) a structured review of each state's childcare licensing and training websites for childcare providers; and (3) in-depth, semi-structured interviews with 31 childcare licensing administrators and technical assistance providers across 17 states. Implementation strategies for supporting childcare providers in adopting healthy eating, physical activity, and screentime regulations vary substantially by state, in quantity and structure. Childcare programs' financial challenges, staff turnover, and lack of adequate facilities were identified as key barriers to adoption. Access to federal food programs was seen as critical to implementing nutrition regulations. Implementation resources such as training and informational materials were rarely available in multiple languages or targeted to providers serving low-income or racially/ethnically diverse families. There is a substantial need for implementation supports for ensuring policies are successfully and equitably implemented in childcare.

In the United States, low-income, underserved rural and urban settings experience poor access to healthy, affordable food. Introducing new food outlets in these locations has shown mixed results for improving healthy food consumption. The Healthy Community Stores Case Study Project (HCSCSP) explored an alternative strategy: supporting mission-driven, locally owned, healthy community food stores to improve healthy food access. The HCSCSP used a multiple case study approach, and conducted a cross-case analysis of seven urban healthy food stores across the United States. The main purpose of this commentary paper is to summarize the main practice strategies for stores as well as future directions for researchers and policy-makers based on results from the prior cross-case analyses. We organize these strategies using key concepts from the Retail Food Environment and Customer Interaction Model. Several key strategies for store success are presented including the use of non-traditional business models, focus on specific retail actors such as store champions and multiple vendor relationships, and a stores' role in the broader community context, as well as the striking challenges faced across store locations. Further exploration of these store strategies and how they are implemented is needed, and may inform policies that can support these types of healthy retail sites and sustain their efforts in improving healthy food access in their communities.

Mission-driven, independently-owned community food stores have been identified as a potential solution to improve access to healthy foods, yet to date there is limited information on what factors contribute to these stores' success and failure. Using a multiple case study approach, this study examined what makes a healthy community food store successful and identified strategies for success in seven community stores in urban areas across the United States. We used Stake's multiple case study analysis approach to identify the following key aims that contributed to community store success across all cases: (1) making healthy food available, (2) offering healthy foods at affordable prices, and (3) reaching community members with limited economic resources. However, stores differed in terms of their intention, action, and achievement of these aims. Key strategies identified that enabled success included: (1) having a store champion, (2) using nontraditional business strategies, (3) obtaining innovative external funding, (4) using a dynamic sourcing model, (5) implementing healthy food marketing, and (6) engaging the community. Stores did not need to implement all strategies to be successful, however certain strategies, such as having a store champion, emerged as critical for all stores. Retailers, researchers, philanthropy, and policymakers can utilize this definition of success and the identified strategies to improve healthy food access in their communities.

Community engagement is well established as a key to improving public health. Prior food environment research has largely studied community engagement as an intervention component, leaving much unknown about how food retailers may already engage in this work. The purpose of this study was to explore the community engagement activities employed by neighborhood food retailers located in lower-income communities with explicit health missions to understand the ways stores involve and work with their communities. A multiple case study methodology was utilized among seven retailers in urban U.S. settings, which collected multiple sources of data at each retailer, including in-depth interviews, store manager sales reports, store observations using the Nutrition Environment Measures Survey for Stores, public documents, and websites. Across-case analysis was performed following Stake's multiple case study approach. Results indicated that retailers employed a wide variety of forms of community engagement within their communities, including Outreach, Building Relationships through Customer Relations, Giving Back, Partnering with Community Coalitions, and Promoting Community Representation and Inclusiveness. Strategies that built relationships through customer relations were most common across stores; whereas few stores demonstrated community inclusiveness where members participated in store decision making. Findings provide a more comprehensive view of the ways local food retailers aim to develop and sustain authentic community relationships. Additional research is needed to evaluate the impact of community engagement activities on improving community health.

Improving healthy food access in low-income communities continues to be a public health challenge. One strategy for improving healthy food access has been to introduce community food stores, with the mission of increasing healthy food access; however, no study has explored the experiences of different initiatives and models in opening and sustaining healthy food stores. This study used a case study approach to understand the experiences of healthy food stores in low-income communities. The purpose of this paper is to describe the methodology used and protocol followed. A case study approach was used to describe seven healthy food stores across urban settings in the U.S. Each site individually coded their cases, and meetings were held to discuss emerging and cross-cutting themes. A cross-case analysis approach was used to produce a series of papers detailing the results of each theme. Most case studies were on for-profit, full-service grocery stores, with store sizes ranging from 900 to 65,000 square feet. Healthy Food Availability scores across sites ranged from 11.6 (low) to 26.5 (high). The papers resulting from this study will detail the key findings of the case studies and will focus on the challenges, strategies, and experiences of retail food stores attempting to improve healthy food access for disadvantaged communities. The work presented in this special issue will help to advance research in the area of community food stores, and the recommendations can be used by aspiring, new, and current community food store owners.

Achieving net-zero CO 2 emissions has become the explicitgoal of many climate-energy policies around the world. Although many studies have assessed net-zero emissions pathways, the common features and tradeoffs of energy systems across global scenarios at the point of net-zero CO 2 emissions have not yet been evaluated. Here, we examine the energy systems of 177 net-zero scenarios and discuss their long-term technological and regional characteristics in the context of current energy policies. We find that, on average, renewable energy sources account for 60% of primary energy at net-zero (compared to ∼14% today), with slightly less than half of that renewable energy derived from biomass. Meanwhile, electricity makes up approximately half of final energy consumed (compared to ∼20% today), highlighting the extent to which solid, liquid, and gaseous fuels remain prevalent in the scenarios even when emissions reach net-zero. Finally, residual emissions and offsetting negative emissions are not evenly distributed across world regions, which may have important implications for negotiations on burden-sharing, human development, and equity., (© 2021. The Author(s).)

Background: Limited clinical data exist describing the use of direct-acting oral anticoagulants (DOACs) in patients with body mass index (BMI) >40 kg/m 2 or body weight >120 kg. Thus, DOAC therapy in this population remains controversial. Objectives: To investigate rivaroxaban as a safe and effective alternative to warfarin for venous thromboembolism (VTE) treatment and prevention of stroke in patients with atrial fibrillation identified as extremely obese or of high body weight. Methods: A retrospective chart review was performed at 2 academic medical centers in patients ≥18 years old and BMI >40 kg/m 2 or weight >120 kg, newly initiated on warfarin or rivaroxaban for atrial fibrillation or VTE treatment. The primary end point was incidence of clinical failure, defined as VTE recurrence, stroke incidence, and mortality, within 12 months of initiation. Secondary end points included length of stay (LOS) and bleeding complications. Results: A total of 176 patients were included, with 84 and 92 patients in the rivaroxaban and warfarin arms, respectively. Clinical failure was lower in the rivaroxaban group but did not reach statistical significance when compared with warfarin (5% vs 13%; P = 0.06). LOS was significantly shorter in the rivaroxaban arm (2 days [1-3] vs 4 days [2-7], P < 0.0001). Percentage of bleeding complications was higher in the rivaroxaban arm but not statistically significant (8% vs 2%, P = 0.06). Conclusion and Relevance: Although not statistically significant, rivaroxaban trended toward a lower incidence of clinical failure while demonstrating a significantly shorter LOS when compared with warfarin for VTE treatment or atrial fibrillation in morbidly obese or high-body-weight patients.

Purpose: Results of a comparison of blood product use and cost outcomes with use of 3-factor versus 4-factor prothrombin complex concentrate (PCC) for indications other than warfarin reversal are presented., Methods: Consecutive patients who received 3-factor PPC (PCC3) or 4-factor PCC (PCC4) for non-warfarin-related indications at 2 U.S. hospitals during a 19-month period were identified. The primary outcome was in-hospital blood product use, with a focus on plasma use. Total hemostasis costs, intensive care unit (ICU) and hospital lengths of stay, and other outcomes were evaluated., Results: Indications for PCC3 use ( n = 118) or PCC4 use ( n = 64) included intraoperative bleeding, nonintraoperative bleeding, coagulopathy of liver disease, and reversal of direct-acting oral anticoagulant effects. The proportion of patients who received plasma was 56.8% with PCC3 use versus 53.1% with PCC4 use ( p = 0.643); the corresponding median volumes of plasma received were 638 mL (interquartile range [IQR], 550-1,355 mL) and 656 mL (IQR, 532-1,136 mL), respectively. The median total hemostasis costs were $5,559 (IQR, $3,922-$8,159) with PCC3 use and $7,771 (IQR, $6,366-$9,205) with PCC4 use ( p < 0.001)., Conclusion: PCC3 use and PCC4 use were associated with similar blood product use, ICU length of stay, hospital length of stay, and in-hospital mortality when given for non-warfarin-related indications. However, relative to PCC3 use, PCC4 use was associated with an increase in costs that was primarily due to drug costs., Competing Interests: DisclosuresDr. Patanwala has previously received grant funding from CSL Behring; he has declared no other potential conflicts of interest. The other authors have declared no potential conflicts of interest., (Copyright © 2018 by the American Society of Health-System Pharmacists, Inc. All rights reserved.)

Background: Prothrombin complex concentrates (PCCs) are drug products containing varying amounts of vitamin K-dependent coagulation factors II, VII, IX, and X. The evidence comparing 3-factor PCC (3-PCC) versus 4-factor PCC (4-PCC) for warfarin reversal is conflicting. It has been hypothesized that 3-PCC may be less effective than 4-PCC because of relatively lower factor VII content., Study Question: The primary objective of this study was to compare international normalized ratio (INR) reversal between 3-PCC and 4-factor PCC (4-PCC) in warfarin-treated patients. The secondary objectives include comparing blood product use, total reversal costs, and cost-effectiveness between the groups., Study Design: This was a retrospective cohort study conducted in 2 affiliated, academic institutions in the United States. Consecutive adult patients who received 3-PCC or 4-PCC for warfarin reversal were included., Measures and Outcomes: The primary outcome was adequate INR reversal defined as a final INR ≤1.5. Secondary outcomes were the utilization of plasma, red blood cells and platelets, reversal costs, and the cost-effectiveness ratio., Results: There were 89 patients who were included in the overall cohort (3-PCC = 57, 4-PCC = 32). Adequate INR reversal occurred less commonly with 3-PCC (45.6%) compared with 4-PCC (87.5%) (P < 0.001). There was no significant difference in the proportion of patients who received plasma (32% vs. 28%, P = 0.813), red blood cells (37% vs. 47%, P = 0.377), or platelets (16% vs. 28%, P = 0.180) between the 3-PCC and 4-PCC groups, respectively. The median reversal cost of 3-PCC ($3663) was lower than 4-PCC ($5105) (P = 0.001). The cost-effective ratio favored 4-PCC ($5105/87.5% = $5834) compared with 3-PCC ($3663/45.6% = $8033)., Conclusions: Four-PCC was more effective than 3-PCC with regard to INR reversal in patients taking warfarin, but blood product use was similar. Although 4-PCC is associated with increased reversal costs, it may be cost-effective in terms of INR reversal.

Objective: To compare the effectiveness and safety of the hemoglobin-based nitric oxide scavenger, pyridoxalated hemoglobin polyoxyethylene, against placebo in patients with vasopressor-dependent distributive shock., Design: Multicenter, randomized, placebo-controlled, open-label study., Setting: Sixty-one participating ICUs in six European countries (Austria, Belgium, Germany, the Netherlands, Spain, and United Kingdom)., Patients: All patients admitted with distributive shock, defined as the presence of at least two systemic inflammatory response syndrome criteria, persisting norepinephrine dependence and evidence of organ dysfunction/hypoperfusion despite adequate fluid resuscitation., Interventions: Patients were randomized to receive 0.25 mL/kg/hr pyridoxalated hemoglobin polyoxyethylene (20 mg Hb/kg/hr) or an equal volume of placebo, infused for up to 150 hours, in addition to conventional vasopressor therapy., Measurements and Main Results: The study was stopped after interim analysis showed higher mortality in the pyridoxalated hemoglobin polyoxyethylene group and an increased prevalence of adverse events. At this time, 377 patients had been randomized to pyridoxalated hemoglobin polyoxyethylene (n = 183) or placebo (n = 194). Age, gender, type of patient (medical/surgical), and Acute Physiology and Chronic Health Evaluation II scores were similar between groups. Twenty-eight-day mortality rate was 44.3% in the pyridoxalated hemoglobin polyoxyethylene group versus 37.6% in the placebo group (OR, 1.29; 95% CI, 0.85-1.95; p = 0.227). In patients with higher organ dysfunction scores (Sepsis-related Organ Failure Assessment > 13), mortality rates were significantly higher in the pyridoxalated hemoglobin polyoxyethylene group when compared with those in placebo-treated patients (60.9% vs 39.2%; p = 0.014). Survivors who received pyridoxalated hemoglobin polyoxyethylene had a longer vasopressor-free time (21.3 vs 19.7 d; p = 0.035)., Conclusions: In this randomized, controlled phase III trial in patients with vasopressor-dependent distributive shock, administration of a pyridoxalated hemoglobin solution decreased the need for vasopressors but was associated with a trend to increased mortality.

It has been half a century since investigators first began experimenting with adding ion exchange resins during the fermentation of microbial natural products. With the development of nonionic polymeric adsorbents in the 1970s, the application of in situ product adsorption in bioprocessing has grown slowly, but steadily. To date, in situ product adsorption strategies have been used in biotransformations, plant cell culture, the production of biofuels, and selected bulk chemicals, such as butanol and lactic acid, as well as in more traditional natural product fermentation within the pharmaceutical industry. Apart from the operational gains in efficiency from the integration of fermentation and primary recovery, the addition of adsorbents during fermentation has repeatedly demonstrated the capacity to significantly increase titers by sequestering the product and preventing or mitigating degradation, feedback inhibition and/or cytotoxic effects. Adoption of in situ product adsorption has been particularly valuable in the early stages of natural product-based drug discovery programs, where quickly and cost-effectively generating multigram quantities of a lead compound can be challenging when using a wild-type strain and fermentation conditions that have not been optimized. While much of the literature involving in situ adsorption describes its application early in the drug development process, this does not imply that the potential for scale-up is limited. To date, commercial-scale processes utilizing in situ product adsorption have reached batch sizes of at least 30,000 l. Here we present examples where in situ product adsorption has been used to improve product titers or alter the ratios among biosynthetically related natural products, examine some of the relevant variables to consider, and discuss the mechanisms by which in situ adsorption may impact the biosynthesis of microbial natural products.

Interleukin-2 (IL-2), a cytokine that induces natural killer cells termed lymphokine-activated killer (LAK) cells, is in use as an anticancer agent. During IL-2 therapy, adverse effects, such as vasodilatation and hypotension, are common. Previous studies suggest that these effects are due to nitric oxide (NO). Therefore a model of IL-2-induced hyperdynamic response in sheep was developed to test the effect of pyridoxalated haemoglobin polyoxyethylene conjugate (PHP; a NO scavenger), which is currently in clinical development for the treatment of shock associated with systemic inflammatory response syndrome. Twelve female sheep were divided into four groups (n =3 per group): sham control (Ringer's lactate solution), PHP alone (20 mg x kg(-1) x h(-1) for 96 h), IL-2 alone (recombinant human IL-2; 1,440,000 units/kg intravenously every 8 h) and a combination of PHP and IL-2. All of the sheep received Ringer's lactate solution to maintain haematocrit at baseline levels. The sheep had free access to food and water. A fall in the mean arterial pressure and systemic vascular resistance index by 20% was observed in the IL-2 group, but not in the PHP+IL-2 group. The fluid requirement to maintain the haematocrit was higher in the IL-2 group (5 ml x kg(-1) x h(-1)) than in the PHP+IL-2 group (4 ml x kg(-1) x h(-1)). The sham group showed no changes in any of the parameters. Scavenging NO by PHP prevented the hyperdynamic reaction induced by IL-2 administration in sheep. This activity of PHP may prevent the early discontinuation of IL-2 therapy that results because of these adverse events.

Redox regulation has been perceived as a simple on-off switch in proteins (corresponding to reduced and oxidized states). Using the transcription factor OxyR as a model, we have generated, in vitro, several stable, posttranslational modifications of the single regulatory thiol (SH), including S-NO, S-OH, and S-SG, and shown that each occurs in vivo. These modified forms of OxyR are transcriptionally active but differ in structure, cooperative properties, DNA binding affinity, and promoter activities. OxyR can thus process different redox-related signals into distinct transcriptional responses. More generally, our data suggest a code for redox control through which allosteric proteins can subserve either graded (cooperative) or maximal (noncooperative) responses, and through which differential responsivity to redox-related signals can be achieved.

We examined the effect of a nitric oxide (NO) quencher, stroma-free human hemoglobin A (HbA0; 0.01, 0.05, 0.1, 0.2 g/kg), on the blood flow measured using the Doppler flow technique, tumor oxygen pressure (pO2) and the diameter of the arterioles using R3230Ac mammary adenocarcinoma as the tumor model. In female Fischer 344 rats with 1-cm-diameter tumors implanted in the lateral aspect of the left quadriceps, intravenous infusion of 0.1 and 0.2 g/kg HbA0 decreased both central tumor and peripheral tumor blood flow by 20-30% (P < 0.05). Tumor pO2 decreased 28% with 0.2 g/kg HbA0, from 15 mm Hg (baseline) to 11 mm Hg at 10 min (P = 0.02). Although 0.2 g/kg HbA0 increased blood flow 55% in the left quadriceps muscle proximal to the implanted tumor (P < 0.05), HbA0 had little effect on blood flow in right quadriceps muscle with no tumor implanted, and increased right quadriceps pO2, from 21 mm Hg (baseline) to 23 mm Hg at 10 min (P = 0.03). HbA0 increased mean arterial pressure 5-10% in a manner that was dependent on dose while heart rate concurrently decreased 9-19%. The diameter of the arterioles supplying the tumor was rapidly reduced 10% by 0.2 g/kg HbA0 (P = 0.037) and remained stable through 60 min of observation (P = 0.005). HbA0 selectively reduces tumor blood flow and tumor pO2 through vasoconstriction of the arterioles supplying the tumor. Vascular NO quenching provides an alternative to NO synthase inhibition as a means to achieve the goal of selective tumor hypoxia.

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the beta 145 tyrosine is substituted with cysteine. The alpha and beta Rainier globin cDNAs were cloned in a high copy number vector and expressed in Saccharomyces cerevisiae under the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual alpha and beta Rainier globin chains. Coexpression of both alpha and beta Rainier cDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the alpha and beta chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A0. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.

Hydrogen peroxide (H2O2) imposes an oxidative stress to Escherichia coli that is manifested by oxidation of glutathione and related redox-sensitive targets. OxyR is a thiol-containing transcriptional activator whose oxidation controls the expression of genes involved in H2O2 detoxification. Here we report that certain S-nitrosothiols (RSNOs) impose what we term a "nitrosative stress" to E. coli, evidenced by lowering of intracellular thiol and the transcriptional activation of OxyR by S-nitrosylation. This cellular and genetic response determines the metabolic fate of RSNOs and thereby contributes to bacterial rescue from stasis. Our studies reveal that signaling by S-nitrosylation can extend to the level of transcription and describe a metabolic pathway that constitutes an adaptation to nitrosative stress.

Hemoglobin (Hbg) and lysates of red blood cells act as vasoconstrictors in isolated vessels by a mechanism that may involve nitric oxide (NO.) scavenging. To determine if such a mechanism indeed occurs, we investigated the effects of cell-free Hgb, modified Hgp (met Hgb and cyan met Hgb), and red blood cells on the formation of NO. induced by endotoxin in rat aorta. Incubation of rat aortic rings with endotoxin induced a delayed and prolonged release of NO. that resulted in a decrease in the contractile response to phenylephrine. Hgb significantly potentiated contractions to phenylephrine in control rings and also reversed the hyporeactivity to this alpha 1-agonist in endotoxin-treated rings with and without endothelium. Lysed red blood cells but not whole red blood cells shifted the concentration-contraction response curves to phenylephrine significantly to the right in endotoxin-treated preparations. Neither picket-fence porphyrin-albumin (PFP-albumin) or metheme-albumin affected the contractile response to phenylephrine. Oxidation of Hgb to met Hgb did not alter the contractions to an alpha 1-agonist in endotoxin-treated rings. In contrast, the formation of cyan met Hgb abolished the action of Hgb on the vascular reactivity of endotoxin-treated preparations. Together, these results demonstrate that free Hgb can scavenge NO. produced in endotoxin-treated vascular tissue and that the ability to bind NO. requires a cell-free form of Hgb with an intact heme center capable of undergoing redox reactions.

Several temperature-sensitive lysis mutants of Saccharomyces cerevisiae were selected according to their ability to release alkaline phosphatase when incubated at a nonpermissive temperature. For two mutants, cell lysis and release of alkaline phosphatase reached a maximum when cells in the logarithmic growth phase were shifted to the nonpermissive temperature. Morphological changes, as well as changes in macromolecular composition of the cells, were observed. Growth is necessary and oxygen is important for the expression of cell lysis at the nonpermissive temperature.