The Mediator kinase module enhances polymerase activity to regulate transcriptional memory after heat stress in Arabidopsis.

Plants are often exposed to recurring adverse environmental conditions in the wild. Acclimation to high temperatures entails transcriptional responses, which prime plants to better withstand subsequent stress events. Heat stress (HS)-induced transcriptional memory results in more efficient re-induction of transcription upon recurrence of heat stress. Here, we identified CDK8 and MED12, two subunits of the kinase module of the transcription co-regulator complex, Mediator, as promoters of heat stress memory and associated histone modifications in Arabidopsis. CDK8 is recruited to heat-stress memory genes by HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2). Like HSFA2, CDK8 is largely dispensable for the initial gene induction upon HS, and its function in transcriptional memory is thus independent of primary gene activation. In addition to the promoter and transcriptional start region of target genes, CDK8 also binds their 3'-region, where it may promote elongation, termination, or rapid re-initiation of RNA polymerase II (Pol II) complexes during transcriptional memory bursts. Our work presents a complex role for the Mediator kinase module during transcriptional memory in multicellular eukaryotes, through interactions with transcription factors, chromatin modifications, and promotion of Pol II efficiency.

Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis.

BACKGROUND: Transcriptional regulation is a key aspect of environmental stress responses. Heat stress induces transcriptional memory, i.e., sustained induction or enhanced re-induction of transcription, that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crop plants is an important breeding goal. However, not all heat stress-inducible genes show transcriptional memory, and it is unclear what distinguishes memory from non-memory genes. To address this issue and understand the genome and epigenome architecture of transcriptional memory after heat stress, we identify the global target genes of two key memory heat shock transcription factors, HSFA2 and HSFA3, using time course ChIP-seq. RESULTS: HSFA2 and HSFA3 show near identical binding patterns. In vitro and in vivo binding strength is highly correlated, indicating the importance of DNA sequence elements. In particular, genes with transcriptional memory are strongly enriched for a tripartite heat shock element, and are hallmarked by several features: low expression levels in the absence of heat stress, accessible chromatin environment, and heat stress-induced enrichment of H3K4 trimethylation. These results are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. CONCLUSIONS: Our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants, enabling the prediction and engineering of genes with transcriptional memory behavior.

Specific functions for Mediator complex subunits from different modules in the transcriptional response of Arabidopsis thaliana to abiotic stress.

Adverse environmental conditions are detrimental to plant growth and development. Acclimation to abiotic stress conditions involves activation of signaling pathways which often results in changes in gene expression via networks of transcription factors (TFs). Mediator is a highly conserved co-regulator complex and an essential component of the transcriptional machinery in eukaryotes. Some Mediator subunits have been implicated in stress-responsive signaling pathways; however, much remains unknown regarding the role of plant Mediator in abiotic stress responses. Here, we use RNA-seq to analyze the transcriptional response of Arabidopsis thaliana to heat, cold and salt stress conditions. We identify a set of common abiotic stress regulons and describe the sequential and combinatorial nature of TFs involved in their transcriptional regulation. Furthermore, we identify stress-specific roles for the Mediator subunits MED9, MED16, MED18 and CDK8, and putative TFs connecting them to different stress signaling pathways. Our data also indicate different modes of action for subunits or modules of Mediator at the same gene loci, including a co-repressor function for MED16 prior to stress. These results illuminate a poorly understood but important player in the transcriptional response of plants to abiotic stress and identify target genes and mechanisms as a prelude to further biochemical characterization.

Victim, Allegation, and Investigation Characteristics Associated With Substantiated Reports of Sexual Abuse of Adults in Residential Care Settings.

The purpose of this study was to identify characteristics of investigations of sexual abuse concerning vulnerable adults residing in facility settings that were associated with case substantiation. Data on 410 reports of sexual abuse were collected prospectively from Adult Protective Services (APS) and state licensure agency staff in New Hampshire, Oregon, Tennessee, Texas, and Wisconsin over a six-month period. Specifically, we examined differences between reports that were substantiated and those that were not by comparing characteristics of alleged victims, alleged perpetrators, and aspects of investigation using logistic regression. We found that a relatively low proportion of cases (18%) were substantiated overall. Compared to cases that were not substantiated, cases that were substantiated were more likely to feature nursing home residents, older victims, female victims, and allegations of physical contact between the alleged perpetrator and victim. Despite the high proportion of alleged perpetrators who were facility staff (51%) compared to resident perpetrators (25%), cases with resident-to-resident allegations of abuse were much more likely to be substantiated, accounting for 63% of substantiated cases. In light of these findings, we believe it is important that investigators are trained to handle sexual abuse cases appropriately and that they are able to investigate the case thoroughly, promptly, and with as much information as possible. It is also critical that investigators make substantiation decisions using the appropriate standard for confirmation (e.g., preponderance of the evidence, beyond a reasonable doubt, clear and convincing evidence) as state law dictates.

Progression of Gastric Acid Production in Preterm Neonates: Utilization of In-vitro Method.

Background: Limited studies are done regarding ability to produce gastric acid in preterm infants and most studies used in vivo method of assessing gastric pH. Objectives: To assess the feasibility of using an in vitro method of measuring gastric pH in babies ≤ 28 weeks gestational age (GA) and determine whether changes in gastric pH differ with gestational age, mode of delivery, and use of antenatal steroids. Design/Methods: Prospective study that enrolled extremely low birth weight (ELBW) babies. Gastric aspirate collected before feeding. In vitro testing of gastric aspirates for pH were done on days of life 1, 3, 5, 7, 14, and 28 by using pH electrode. The pH was measured on each sample in triplicate, mean calculated and used for data analysis. Stastical methods included descriptive statistics, t-tests and repeated measures ANOVA. Results: 29 subjects ≤ 28 weeks or birth weight ≤ 1,000 g were enrolled. No significant change was noted in pH measurements over time. Antenatal steroids and mode of delivery did not affect gastric acid pH. Conclusion: The in vitro method for gastric pH measurements is non-invasive and affords more frequent testing. It would be useful in studying various conditions that may affect gastric pH.

Redox regulation of PEP activity during seedling establishment in Arabidopsis thaliana.

Activation of the plastid-encoded RNA polymerase is tightly controlled and involves a network of phosphorylation and, as yet unidentified, thiol-mediated events. Here, we characterize PLASTID REDOX INSENSITIVE2, a redox-regulated protein required for full PEP-driven transcription. PRIN2 dimers can be reduced into the active monomeric form by thioredoxins through reduction of a disulfide bond. Exposure to light increases the ratio between the monomeric and dimeric forms of PRIN2. Complementation of prin2-2 with different PRIN2 protein variants demonstrates that the monomer is required for light-activated PEP-dependent transcription and that expression of the nuclear-encoded photosynthesis genes is linked to the activity of PEP. Activation of PEP during chloroplast development likely is the source of a retrograde signal that promotes nuclear LHCB expression. Thus, regulation of PRIN2 is the thiol-mediated mechanism required for full PEP activity, with PRIN2 monomerization via reduction by TRXs providing a mechanistic link between photosynthetic electron transport and activation of photosynthetic gene expression.

The role of retrograde signals during plant stress responses.

Chloroplast and mitochondria not only provide the energy to the plant cell but due to the sensitivity of organellar processes to perturbations caused by abiotic stress, they are also key cellular sensors of environmental fluctuations. Abiotic stresses result in reduced photosynthetic efficiency and thereby reduced energy supply for cellular processes. Thus, in order to acclimate to stress, plants must re-program gene expression and cellular metabolism to divert energy from growth and developmental processes to stress responses. To restore cellular energy homeostasis following exposure to stress, the activities of the organelles must be tightly co-ordinated with the transcriptional re-programming in the nucleus. Thus, communication between the organelles and the nucleus, so-called retrograde signalling, is essential to direct the energy use correctly during stress exposure. Stress-triggered retrograde signals are mediated by reactive oxygen species and metabolites including ß-cyclocitral, MEcPP (2-C-methyl-d-erythritol 2,4-cyclodiphosphate), PAP (3'-phosphoadenosine 5'-phosphate), and intermediates of the tetrapyrrole biosynthesis pathway. However, for the plant cell to respond optimally to environmental stress, these stress-triggered retrograde signalling pathways must be integrated with the cytosolic stress signalling network. We hypothesize that the Mediator transcriptional co-activator complex may play a key role as a regulatory hub in the nucleus, integrating the complex stress signalling networks originating in different cellular compartments.

Impact of Mother-Daughter Relationship on Hypertension Self-management and Quality of Life: Testing Dyadic Dynamics Using the Actor-Partner Interdependence Model.

BACKGROUND: Although hypertension (HTN) treatment rates are similar across age groups of women, effective control is significantly worse among older women. Only 20% of hypertensive women aged 70 to 79 years have controlled blood pressure. OBJECTIVES: The purpose of this longitudinal study was to test the effects of the quality of mother-daughter relationship, inner strength, and control on HTN self-management and health-related quality of life (HRQOL) for both members of the dyad at 6 months. The Actor-Partner Interdependence Model was used to examine the direct ("actor") and indirect ("partner") effects of 46 dyads. RESULTS: The mothers' perceived relationship quality with daughters directly impacted their own self-management of HTN and HRQOL while also indirectly affecting their daughters' self-management. Similarly, the daughters' perceived strength of their relationship with their mothers directly influenced their self-management and HRQOL and indirectly affected their mothers' self-management and HRQOL.

Mutation of Gly195 of the ChlH Subunit of Mg-chelatase Reduces Chlorophyll and Further Disrupts PS II Assembly in a Ycf48-Deficient Strain of Synechocystis sp. PCC 6803.

Biogenesis of the photosystems in oxygenic phototrophs requires co-translational insertion of chlorophyll a. The first committed step of chlorophyll a biosynthesis is the insertion of a Mg(2+) ion into the tetrapyrrole intermediate protoporphyrin IX, catalyzed by Mg-chelatase. We have identified a Synechocystis sp. PCC 6803 strain with a spontaneous mutation in chlH that results in a Gly195 to Glu substitution in a conserved region of the catalytic subunit of Mg-chelatase. Mutant strains containing the ChlH Gly195 to Glu mutation were generated using a two-step protocol that introduced the chlH gene into a putative neutral site in the chromosome prior to deletion of the native gene. The Gly195 to Glu mutation resulted in strains with decreased chlorophyll a. Deletion of the PS II assembly factor Ycf48 in a strain carrying the ChlH Gly195 to Glu mutation did not grow photoautotrophically. In addition, the ChlH-G195E:ΔYcf48 strain showed impaired PS II activity and decreased assembly of PS II centers in comparison to a ΔYcf48 strain. We suggest decreased chlorophyll in the ChlH-G195E mutant provides a background to screen for the role of assembly factors that are not essential under optimal growth conditions.

Comparison of D1´- and D1-containing PS II reaction centre complexes under different environmental conditions in Synechocystis sp. PCC 6803.

In oxygenic photosynthesis, the D1 protein of Photosystem II is the primary target of photodamage and environmental stress can accelerate this process. The cyanobacterial response to stress includes transcriptional regulation of genes encoding D1, including low-oxygen-induction of psbA1 encoding the D1´ protein in Synechocystis sp. PCC 6803. The psbA1 gene is also transiently up-regulated in high light, and its deletion has been reported to increase ammonium-induced photoinhibition. Therefore we investigated the role of D1´-containing PS II centres under different environmental conditions. A strain containing only D1´-PS II centres under aerobic conditions exhibited increased sensitivity to ammonium chloride and high light compared to a D1-containing strain. Additionally a D1´-PS II strain was outperformed by a D1-PS II strain under normal conditions; however, a strain containing low-oxygen-induced D1´-PS II centres was more resilient under high light than an equivalent D1 strain. These D1´-containing centres had chlorophyll a fluorescence characteristics indicative of altered forward electron transport and back charge recombination with the donor side of PS II. Our results indicate D1´-PS II centres are important in the reconfiguration of thylakoid electron transport in response to high light and low oxygen.

Environmental pH affects photoautotrophic growth of Synechocystis sp. PCC 6803 strains carrying mutations in the lumenal proteins of PSII.

Synechocystis sp. strain PCC 6803 grows photoautotrophically across a broad pH range, but wild-type cultures reach a higher density at elevated pH; however, photoheterotrophic growth is similar at high and neutral pH. A number of PSII mutants each lacking at least one lumenal extrinsic protein, and carrying a second PSII lumenal mutation, are able to grow photoautotrophically in BG-11 medium at pH 10.0, but not pH 7.5. We investigated the basis of this pH effect and observed no pH-specific change in variable fluorescence yield from PSII centers of the wild type or the pH-dependent ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains; however, 77 K fluorescence emission spectra indicated increased coupling of the phycobilisome (PBS) antenna at pH 10.0 in all mutants. DNA microarray data showed a cell-wide response to transfer from pH 10.0 to pH 7.5, including decreased mRNA levels of a number of oxidative stress-responsive transcripts. We hypothesize that this transcriptional response led to increased tolerance against reactive oxygen species and in particular singlet oxygen. This response enabled photoautotrophic growth of the PSII mutants at pH 10.0. This hypothesis was supported by increased resistance of all strains to rose bengal at pH 10.0 compared with pH 7.5.

Association of intimate partner violence and childhood sexual abuse with cancer-related well-being in women.

BACKGROUND: Limited evidence suggests that intimate partner violence (IPV) may be associated with poorer cancer outcomes. We hypothesized that timing and type of IPV as well as childhood sexual abuse (CSA) may negatively affect depression, perceived stress, and cancer-related well-being. METHODS: This was a cross-sectional study of women diagnosed with either breast, cervical, or colorectal cancer in the prior 12 months included in the Kentucky Cancer Registry. Consenting women were interviewed by phone (n=553). Multivariate analysis of covariance (MANCOVA) was used to determine the association between IPV (37% lifetime prevalence) and type, timing, and the range of correlated cancer-related well-being indicators, adjusting for confounding factors. RESULTS: IPV (p=0.002) and CSA (p=0.03) were associated with the six correlated well-being indicators. Specifically, lifetime and current IPV were associated with lower Functional Assessment of Cancer Therapy-Breast Cancer (FACT-B) (p=0.006) and Functional Assessment of Chronic Illness Therapy-Spiritual Well-being Scale (FACIT-SP) (p=0.03) scores, higher perceived stress at diagnosis (p=0.006), and depressive symptom scores at diagnosis (p<0.0001), whereas CSA was associated with lower FACT-B (p=0.02), increased number of comorbid conditions (p=0.03), and higher current stress levels (p=0.04). Current and past IPV, as well as psychologic abuse, were associated with poorer well-being among women with a recent cancer diagnosis. CONCLUSIONS: Our results provide evidence that both IPV and CSA negatively influence cancer-related well-being indicators. These data suggest that identification of lifetime IPV and other stressors may provide information that healthcare providers can use to best support and potentially improve the well-being of female cancer patients.

Universal psychosocial screening and adverse pregnancy outcomes in an academic obstetric clinic.

OBJECTIVE: To test the hypothesis that women screened for psychosocial factors, including partner abuse, as recommended by the American College of Obstetricians and Gynecologists would have higher neonatal birth weight, longer gestational age at delivery, higher Apgar scores, and fewer maternal complications. METHODS: We evaluated a universal psychosocial screening intervention using a retrospective cohort (n=881 prenatal care patients). Pregnancy outcomes among patients screened beginning in 2008 (n=464) were compared with outcomes among women receiving care before universal screening was implemented (n=417). Data were obtained from medical records between 2007 and 2009. Multivariable logistic regression and analysis of covariance were used to estimate the association between screening and pregnancy outcomes among singleton births adjusting for confounders (prior preterm births, insurance, and mode of delivery). RESULTS: Screened women were less likely than women not universally screened to have low birth weight neonates (4.5% of screened, 10.3% of unscreened; adjusted odds ratio [OR] 0.41, 95% confidence interval [CI] 0.23-0.73), preterm births (9.9% of screened, 14.9% of unscreened; adjusted OR 0.62, 95% CI 0.41-0.96), and any maternal complication (30.0% of screened, 41.2% of unscreened; adjusted OR 0.67, 95% CI 0.50-0.88). Newborn Apgar scores were higher (P=.01) among screened relative to unscreened mothers. CONCLUSION: Our results provide evidence that universal screening was associated with improved pregnancy outcomes. LEVEL OF EVIDENCE: II.

Foreign born status and HIV/AIDS: a comparative analysis of HIV/AIDS characteristics among foreign and U.S. born individuals.

The purpose of this study was to observe differences in HIV characteristics between the foreign and U.S. born HIV population. The study sample consisted of individuals, ≥13 years of age, who have been diagnosed with HIV and are patients of the Lexington Bluegrass Care Clinic. For the comparison analysis, the sample was divided into two groups (n = 1070), foreign born and U.S. born. Compared to U.S. born patients, foreign born patients were younger at the time of their HIV diagnosis (31 years vs. 36 years, P = 0.005), of Hispanic origin (63.1% vs. 1.1%, P < 0.001), and reported heterosexual contact as their mode of transmission (67.7% vs. 33.9% P < 0.001). Foreign born patients enrolled into care at a much lower CD4+ count and compared to the U.S. born, they were more likely to be classified as late testers (53.9% vs. 32.8%, P = 0.006). There were many differences found between foreign born and U.S. born HIV patients. Upon enrolling into care, foreign born individuals were more likely to have multiple OI's and have an AIDS diagnosis. In regards to testing, foreign born individuals were more likely to have tested late for HIV compared to their U.S. born counterparts.

An e-Research approach to Web-scale music analysis.

The growing quantity of digital recorded music available in large-scale resources such as the Internet archive provides an important new resource for musical analysis. An e-Research approach has been adopted in order to create a very substantive web-accessible corpus of musical analyses in a common framework for use by music scholars, students and beyond, and to establish a methodology and tooling that will enable others to add to the resource in the future. The enabling infrastructure brings together scientific workflow and Semantic Web technologies with a set of algorithms and tools for extracting features from recorded music. It has been used to deliver a prototype system, described here, that demonstrates the utility of LINKED DATA for enhancing the curation of collections of music signal data for analysis and publishing results that can be simply and readily correlated to these and other sources. This paper describes the motivation, infrastructure design and the proof-of-concept case study and reflects on emerging e-Research practice as researchers embrace the scale of the Web.

Occupational status, work-site cessation programs and policies and menthol smoking on quitting behaviors of US smokers.

AIM: This exploratory study sought to examine the relationships among occupational status, menthol smoking preference and employer-sponsored smoking cessation programs and policies on quitting behaviors. DESIGN: Data for this cross-sectional study were obtained from the 2006 Tobacco Use Supplement to the Current Population Survey (TUS CPS), a large national survey representative of the civilian population, containing approximately 240,000 respondents. The total sample for the current study was 30,176. MEASUREMENTS: The TUS CPS regularly collects data on cigarette prevalence, quitting behaviors, smoking history and consumption patterns. We performed a logistic regression with 'life-time quitting smoking for 1 day or longer because they were trying to quit' as outcome variable. Independent variables included type of occupation, employer-sponsored cessation programs and policies and menthol status. FINDINGS: When controlling for occupational status and work-place policies, there were no differences for menthol versus non-menthol smokers on quitting behaviors [odds ratio (OR) = 0.98; 95% confidence interval (CI) = 0.83, 1.15]. Service workers were less likely to quit compared with white-collar workers (OR = 0.80; 95% CI = 0.69, 0.94), and those with no employer-sponsored cessation program were less likely to quit (OR = 0.70; 95% CI = 0.60, 0.83). White-collar workers, compared with blue-collar and service workers, were more likely to have a smoking policy in the work area (93% versus 86% versus 88%, respectively). CONCLUSIONS: When occupational status and work-place smoking policies are controlled for, smokers of menthol cigarettes in the United States appear to have similar self-reported life-time rates of attempts to stop smoking to non-menthol smokers.

Health profile differences for menthol and non-menthol smokers: findings from the National Health Interview Survey.

AIMS: Although the adverse effects of smoking are well known, limited information exists about the overall health profiles of menthol smokers when compared to their non-menthol smoking counterparts. Using a well-known nationally representative survey, this study examines differences between self-reported health characteristics for menthol and non-menthol smokers. DESIGN: Cross-sectional data from the 2005 National Health Interview Survey and its cancer control supplement were used to analyze responses for current and former smokers (n = 12,004) independently. All analyses were conducted using SAS version 9.2 and SAS callable SUDAAN version 9.0.3. Multiple logistic regression analysis was used to model menthol smoking. FINDINGS: After controlling for sex, age and race, we found that in current smokers the mean number of cigarettes smoked per day is significantly lower for menthol smokers when compared to non-menthol smokers [odds ratio (OR): 0.99; 95% confidence interval (CI): 0.98, 1.00]. Also, we found that former menthol smokers had higher body mass indices (BMIs) (OR: 1.01; 95% CI: 1.00, 1.02) and were more likely to have visited the emergency room due to asthma (OR: 2.30, 95% CI: 1.04, 5.09). CONCLUSIONS: Overall, current menthol and non-menthol smokers have similar health profiles. However, menthol smokers reported smoking fewer cigarettes per day than their non-menthol counterparts. While these findings are supportive of other published data, future studies may need to tease out the health-related significance of smoking fewer menthol cigarettes per day but having similar health outcomes to those who smoke more non-menthol cigarettes per day. Additionally, our findings suggest that there may be some differences between the former menthol and non-menthol smoker.

A scaling approach for quantifying the net CO2 flux of the Kuparuk River Basin, Alaska.

Net CO2 flux measurements conducted during the summer and winter of 1994-96 were scaled in space and time to provide estimates of net CO2 exchange during the 1995-96 (9 May 1995-8 May 1996) annual cycle for the Kuparuk River Basin, a 9200 km2 watershed located in NE Alaska. Net CO2 flux was measured using dynamic chambers and eddy covariance in moist-acidic, nonacidic, wet-sedge, and shrub tundra, which comprise 95% of the terrestrial landscape of the Kuparuk Basin. CO2 flux data were used as input to multivariate models that calculated instantaneous and daily rates of gross primary production (GPP) and whole-ecosystem respiration (R) as a function of meteorology and ecosystem development. Net CO2 flux was scaled up to the Kuparuk Basin using a geographical information system (GIS) consisting of a vegetation map, digital terrain map, dynamic temperature and radiation fields, and the models of GPP and R. Basin-wide estimates of net CO2 exchange for the summer growing season (9 May-5 September 1995) indicate that nonacidic tundra was a net sink of -31.7 ± 21.3 GgC (1 Gg = 109 g), while shrub tundra lost 32.5 ± 6.3 GgC to the atmosphere (negative values denote net ecosystem CO2 uptake). Acidic and wet sedge tundra were in balance, and when integrated for the entire Kuparuk River Basin (including aquatic surfaces), whole basin summer net CO2 exchange was estimated to be in balance (-0.9 ± 50.3 GgC). Autumn to winter (6 September 1995-8 May 1996) estimates of net CO2 flux indicate that acidic, nonacidic, and shrub tundra landforms were all large sources of CO2 to the atmosphere (75.5 ± 8.3, 96.4 ± 11.4, and 43.3 ± 4.7 GgC for acidic, nonacidic, and shrub tundra, respectively). CO2 loss from wet sedge surfaces was not substantially different from zero, but the large losses from the other terrestrial landforms resulted in a whole basin net CO2 loss of 217.2 ± 24.1 GgC during the 1995-96 cold season. When integrated for the 1995-96 annual cycle, acidic (66.4 + 25.25 GgC), nonacidic (64.7 ± 29.2 GgC), and shrub tundra (75.8 ± 8.4 GgC) were substantial net sources of CO2 to the atmosphere, while wet sedge tundra was in balance (0.4 + 0.8 GgC). The Kuparuk River Basin as a whole was estimated to be a net CO2 source of 218.1 ± 60.6 GgC over the 1995-96 annual cycle. Compared to direct measurements of regional net CO2 flux obtained from aircraft-based eddy covariance, the scaling procedure provided realistic estimates of CO2 exchange during the summer growing season. Although winter estimates could not be assessed directly using aircraft measurements of net CO2 exchange, the estimates reported here are comparable to measured values reported in the literature. Thus, we have high confidence in the summer estimates of net CO2 exchange and reasonable confidence in the winter net CO2 flux estimates for terrestrial landforms of the Kuparuk river basin. Although there is larger uncertainty in the aquatic estimates, the small surface area of aquatic surfaces in the Kuparuk river basin (≈ 5%) presumably reduces the potential for this uncertainty to result in large errors in basin-wide CO2 flux estimates.