A Novel Type of Monocytic Leukemia Stem Cell Revealed by the Clinical Use of Venetoclax-Based Therapy.

The BCL2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine metabolism, and selective sensitivity to cladribine. Critically, in some instances, m-LSC and p-LSC subtypes can co-reside in the same patient with AML and simultaneously contribute to overall tumor biology. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlight the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens. SIGNIFICANCE: These studies identify and characterize a new type of human acute myeloid LSC that is responsible for monocytic disease progression in patients with AML treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass. This article is featured in Selected Articles from This Issue, p. 1949.

Longitudinal analysis of SARS-CoV-2 spike and RNA-dependent RNA polymerase protein sequences reveals the emergence and geographic distribution of diverse mutations.

Amid the ongoing COVID-19 pandemic, it has become increasingly important to monitor the mutations that arise in the SARS-CoV-2 virus, to prepare public health strategies and guide the further development of vaccines and therapeutics. The spike (S) protein and the proteins comprising the RNA-Dependent RNA Polymerase (RdRP) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences were downloaded from the GISAID database, aligned, and the variants identified. 437,006 unique viral genomes were analyzed. Polymorphisms in the protein sequence were investigated and examined longitudinally to identify sequence and strain variants appearing between January 5th, 2020 and January 16th, 2021. A structural analysis was also performed to investigate mutations in the receptor binding domain and the N-terminal domain of the spike protein. Within the spike protein, there were 766 unique mutations observed in the N-terminal domain and 360 in the receptor binding domain. Four residues that directly contact ACE2 were mutated in more than 100 sequences, including positions K417, Y453, S494, and N501. Within the furin cleavage site of the spike protein, a high degree of conservation was observed, but the P681H mutation was observed in 10.47% of sequences analyzed. Within the RNA dependent RNA polymerase complex proteins, 327 unique mutations were observed in Nsp8, 166 unique mutations were observed in Nsp7, and 1157 unique mutations were observed in Nsp12. Only 4 sequences analyzed contained mutations in the 9 residues that directly interact with the therapeutic Remdesivir, suggesting limited mutations in drug interacting residues. The identification of new variants emphasizes the need for further study on the effects of the mutations and the implications of increased prevalence, particularly for vaccine or therapeutic efficacy.

Hysteresis analysis of nitrate dynamics in the Neuse River, NC.

Anthropogenic activities have caused N saturation in many terrestrial ecosystems. The transfer of nutrients and sediments to freshwater environments has resulted in water quality impairments including eutrophication, increased turbidity, ecosystem acidification, and loss of biodiversity. Storm events account for the transport of a large proportion of nutrients and sediments found in watersheds on an annual basis. To implement effective water-quality management strategies, the importance of surface and subsurface flow paths during storm events and low flow conditions need to be quantified. The increased availability of optical in-situ sensors makes high-frequency monitoring of catchment fluxes practical. In this study, we present a high-resolution nitrate monitoring record over a 10-year period in the Neuse River Basin near Clayton, North Carolina. The relationship between discharge and nitrate concentration for 365 storm events are categorized into hysteresis classes that indicate different transport mechanisms into the river. Storm events over the entire period of this study are divided between clockwise, counter-clockwise, and complex hysteresis patterns, indicating multiple nitrate flow paths during different seasons and years. Logistic regression of a suite of environmental variables demonstrates that antecedent soil moisture is a significant factor in determining the storm hysteresis class, with the odds of counter-clockwise hysteresis increasing by 10.3% for every 1 percentage point increase in the soil moisture. There is also an overlying seasonal effect, which indicates that dry soil conditions and frequent small storms during summer leads to greater nitrate transport on the rising limb, in contrast to slower, groundwater-driven inputs during the rest of the year.

Back-trajectory analysis and source-receptor relationships: particulate matter and nitrogen isotopic composition in rainwater.

The southeastern portion of North Carolina features a dense crop and animal agricultural region; previous research suggests that this agricultural presence emits a significant portion of the state's nitrogen (i.e., oxides of nitrogen and ammonia) emissions. These findings indicate that transporting air over this region can affect nitrogen concentrations in precipitation at sites as far as 50 mi away. The study combined nitrate nitrogen isotope data with back-trajectory analysis to examine the relationship between regional nitrogen emission estimates independent of pollutant concentration information. In 2004, the Hybrid Single Particle Lagrangian Integrated Trajectory (HYSPLIT) model was used to determine potential sources of nitrogen in rainwater collected at an urban receptor site in Raleigh, NC. The delta 15N isotope ratio signatures of each sample were used to further differentiate between sources of the rainwater nitrate. This study examined the importance of pollution sources, including animal agricultural activity, and meteorology on rainfall chemistry as well as the implications in fine particulate matter (PM2.5) formation. Samples that transited the dense crop and animal (swine) agricultural region of east-southeastern North Carolina (i.e., the source region) had lower delta 15N isotope ratios in the nitrate ion (average = -2.1 +/- 1.7 per thousand) than those from a counterpart nonagricultural region (average = 0.1 +/- 3 per thousand.) An increase in PM2.5 concentrations in the urban receptor site (yearly average = 15.1 +/- 5.8 microg/m3) was also found to correspond to air transport over the dense agricultural region relative to air that was not subjected to such transport (yearly average = 11.7 +/- 5.8 microg/m3).

Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae).

Hemolymph, the circulatory fluid of bivalves, transports nutrients, respiratory gases, enzymes, metabolic wastes, and toxicants throughout the body. Hemolymph can provide information pertinent to health assessment of animals or populations, but is not commonly used in freshwater bivalves partly because of the lack of tested, practical techniques for its nonlethal collection. The objective of this study was to evaluate the effect of hemolymph collection on the growth and survival of Elliptio complanata, a freshwater bivalve (Unionidae). We describe a simple technique for the collection of hemolymph from the anterior adductor muscle sinus of E. complanata. To evaluate the effect of hemolymph sampling on mussel survival and growth, 30 mussels sampled using the technique and 30 unsampled controls were followed for 3 mo post collection. Nine animals were sampled 3 times over 7 mo to monitor effects of repeated sampling. No negative impacts on survival or growth were observed in either the singly or repeatedly sampled animals. We also compared the composition of hemolymph collected from the adductor muscle sinus with that collected from the ventricle of the heart. Calcium levels and cell count of hemolymph obtained from the adductor sinus and ventricle were significantly different. There was no significant difference between collection sites for magnesium, phosphorus, ammonia, protein, sodium, potassium, or chloride. We conclude that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.

Reference ranges for hemolymph chemistries from Elliptio complanata of North Carolina.

Hemolymph chemistries may be useful nonlethal measures of bivalve health. The prognostic value of hemolymph, however, depends on a comparison of chemistry results to reference ranges from healthy individuals. Currently, knowledge of expected hemolymph values in healthy and unhealthy freshwater mussels is extremely limited. The purpose of this study was to develop a set of reference ranges for clinical evaluation of hemolymph from a freshwater mussel species common to southeastern USA. We collected hemolymph from 380 Elliptio complanata from 19 apparently healthy populations from northwest of Raleigh, North Carolina, during May through July 2001. We present reference ranges for hemolymph parameters ammonia, glucose, calcium, magnesium, phosphorus, aspartate aminotransferase (AST), bicarbonate, protein and cell count, and for tissue glycogen. We compare the subpopulations of mussels from regions with an agricultural riparian buffer to those surrounded predominantly by forested lands. We further present correlations noted between hemolymph chemistries and physical or physiologic parameters. The only statistically significant differences between populations contiguous to agricultural and forested lands were in hemolymph calcium and glucose concentrations. Other statistically significant correlations identified were between gravidity and hemolymph protein concentration and tissue glycogen content, as well as between gravidity and parasite burden, and between shell length and hemolymph glucose, AST, calcium and bicarbonate concentrations. The results of this study will aid the interpretation of health measures from populations of E. complanata of similar geographic and seasonal origin.

Nitrate movement in shallow ground water from swine-lagoon-effluent spray fields managed under current application regulations.

Rapid increases in the swine (Sus scrofa domestica) population in the 1990s and associated potential for nitrate N pollution of surface waters led the state of North Carolina to adopt stringent waste management regulations in 1993. Our objectives were to characterize (i) nitrate N movement from waste application fields (WAFs) in shallow ground water, and (ii) soil, hydrologic, and biological factors influencing the amount of nitrate N in the adjacent stream. A ground water monitoring study was conducted for 36 mo on a swine farm managed under new regulations. Water table contours and lack of vertical gradients indicated horizontal flow over most of the site. Nitrate N concentrations in water from shallow wells in WAFs averaged 30 +/- 19 mg L(-1) and delta15N ratios for nitrate N were between +20 and +25 per mil. Nitrate N concentration decreased from field-edge to streamside wells by 22 to 99%. Measurement of delta18O and delta15N enrichment of nitrate in ground water throughout the WAF-riparian system indicated that denitrification has not caused significant 15N enrichment of nitrate. Over a 24-mo period, delta15N ratios for nitrate N in the stream approached delta15N ratios for nitrate N in ground water beneath WAFs indicating delivery of some waste-derived nitrate N to the stream in shallow ground water. Nitrate N concentrations in the stream were relatively low, averaging 1 mg L(-1). Dilution of high nitrate N water in shallow horizontal flow paths with low nitrate N water from deeper horizontal flow paths at or near the stream, some denitrification as ground water discharges through the stream bottom, and some denitrification in riparian zone contributed to this low nitrate N concentration.

Key metabolites in tissue extracts of Elliptio complanata identified using (1)H nuclear magnetic resonance spectroscopy.

We used (1)H nuclear magnetic resonance spectroscopy to describe key metabolites of the polar metabolome of the freshwater mussel, Elliptio complanata. Principal components analysis documented variability across tissue types and river of origin in mussels collected from two rivers in North Carolina (USA). Muscle, digestive gland, mantle and gill tissues yielded identifiable but overlapping metabolic profiles. Variation in digestive gland metabolic profiles between the two mussel collection sites was characterized by differences in mono- and disaccharides. Variation in mantle tissue metabolomes appeared to be associated with sex. Nuclear magnetic resonance spectroscopy is a sensitive means to detect metabolites in the tissues of E. complanata and holds promise as a tool for the investigation of freshwater mussel health and physiology.

Escalation and extinction selectivity: morphology versus isotopic reconstruction of bivalve metabolism.

Studies that have tested and failed to support the hypothesis that escalated species (e.g., those with predation-resistant adaptations) are more susceptible to elimination during mass extinctions have concentrated on the distribution and degree of morphological defenses in molluscan species. This morphological approach to determining level of escalation in bivalves may be oversimplified because it does not account for metabolic rate, which is an important measure of escalation that is less readily accessible for fossils. Shell growth rates in living bivalves are positively correlated with metabolic rate and thus are potential indicators of level of escalation. To evaluate this approach, we used oxygen isotopes to reconstruct shell growth rates for two bivalve species (Macrocallista marylandica and Glossus markoei) from Miocene-aged sediments of Maryland. Although both species are classified as non-escalated based on morphology, the isotopic data indicate that M. marylandica was a faster-growing species with a higher metabolic rate and G. markoei was a slower-growing species with a lower metabolic rate. Based on these results, we predict that some morphologically non-escalated species in previous tests of extinction selectivity should be reclassified as escalated because of their fast shell growth rates (i.e., high metabolic rates). Studies that evaluate the level of escalation of a fauna should take into account the energetic physiology of taxa to avoid misleading results.

Application of classification-tree methods to identify nitrate sources in ground water.

A study was conducted to determine if nitrate sources in ground water (fertilizer on crops, fertilizer on golf courses, irrigation spray from hog (Sus scrofa) wastes, and leachate from poultry litter and septic systems) could be classified with 80% or greater success. Two statistical classification-tree models were devised from 48 water samples containing nitrate from five source categories. Model 1 was constructed by evaluating 32 variables and selecting four primary predictor variables (delta 15N, nitrate to ammonia ratio, sodium to potassium ratio, and zinc) to identify nitrate sources. A delta 15N value of nitrate plus potassium > 18.2 indicated animal sources; a value < 18.2 indicated inorganic or soil organic N. A nitrate to ammonia ratio > 575 indicated inorganic fertilizer on agricultural crops; a ratio < 575 indicated nitrate from golf courses. A sodium to potassium ratio > 3.2 indicated septic-system wastes; a ratio < 3.2 indicated spray or poultry wastes. A value for zinc > 2.8 indicated spray wastes from hog lagoons; a value < 2.8 indicated poultry wastes. Model 2 was devised by using all variables except delta 15N. This model also included four variables (sodium plus potassium, nitrate to ammonia ratio, calcium to magnesium ratio, and sodium to potassium ratio) to distinguish categories. Both models were able to distinguish all five source categories with better than 80% overall success and with 71 to 100% success in individual categories using the learning samples. Seventeen water samples that were not used in model development were tested using Model 2 for three categories, and all were correctly classified. Classification-tree models show great potential in identifying sources of contamination and variables important in the source-identification process.

Nitrate contamination in groundwater on an urbanized dairy farm.

Urbanization of rural farmland is a pervasive trend around the globe, and maintaining and protecting adequate water supplies in suburban areas is a growing problem. Identification of the sources of groundwater contamination in urbanized areas is problematic, but will become important in areas of rapid population growth and development. The isotopic composition of NO3 (delta15N(NO3) and delta18O NO3), NH4 (delta15N(NH4)), groundwater (delta2H(wt) and delta18O(wt)) and chloride/bromide ratios were used to determine the source of nitrate contamination in drinking water wells in a housing development that was built on the site of a dairy farm in the North Carolina Piedmont, U.S. The delta15N(NO3) and delta18O NO3 compositions imply that elevated nitrate levels at this site in drinking well water are the result of waste contamination, and that denitrification has not significantly attenuated the groundwater nitrate concentrations. delta15N(NO3) and delta18O(NO3) compositions in groundwater could not differentiate between septic effluent and animal waste contamination. Chloride/ bromide ratios in the most contaminated drinking water wells were similar to ratios found in animal waste application fields, and were higher than Cl/Br ratios observed in septic drain fields in the area. delta18O(wt) was depleted near the site of a buried waste lagoon without an accompanying shift in delta2H(wt) suggesting water oxygen exchange with CO2. This water-CO2 exchange resulted from the reduction of buried lagoon organic matter, and oxidation of the released gases in aerobic soils. delta18O(wt) is not depleted in the contaminated drinking water wells, indicating that the buried dairy lagoon is not a source of waste contamination. The isotope and Cl/Br ratios indicate that nitrate contamination in these drinking wells are not from septic systems, but are the result of animal waste leached from pastures into groundwater during 35 years of dairy operations which did not violate any existing regulations. Statutes need to be enacted to protect the health of the homeowners that require well water to be tested prior to the sale of homes built on urbanized farmland.

Uncertainties in the oxygen isotopic composition of barium sulfate induced by coprecipitation of nitrate.

Coprecipitation of nitrate and sulfate by barium has probably resulted in significant error in numerous studies dealing with the oxygen isotopic composition of natural sulfates using chemical/thermal conversion of BaSO(4) and analysis by isotope ratio mass spectrometry. In solutions where NO(3) (-)/SO(4) (2-) molar ratios are above 2 the amount of nitrate coprecipitated with BaSO(4) reaches a maximum of approximately 7% and decreases roughly linearly as the molar ratio decreases. The fraction of coprecipitated nitrate appears to increase with decreasing pH and is also affected by the nature of the cations in the precipitating solution. The size of the oxygen isotope artifact in sulfate depends both on the amount of coprecipitated nitrate and the delta(18)O and Delta(17)O values of the nitrate, both of which can be highly variable. The oxygen isotopic composition of sulfate extracted from atmospheric aerosols or rain waters are probably severely biased because photochemical nitrate is usually also present and it is highly enriched in (18)O (delta(18)O approximately 50-90 per thousand) and has a large mass-independent isotopic composition (Delta(17)O approximately 20-32 per thousand). The sulfate delta(18)O error can be 2-5 per thousand with Delta(17)O artifacts reaching as high as 4.0 per thousand.

Temporal and spatial variability in stable isotope compositions of a freshwater mussel: implications for biomonitoring and ecological studies.

Stable isotopes can be used to elucidate ecological relationships in community and trophic studies. Findings are calibrated against baselines, e.g. from a producer or primary consumer, assumed to act as a reference to the isotopic context created by spatio-temporal attributes such as geography, climate, nutrient, and energy sources. The ability of an organism to accurately represent a community base depends on how, and over what time-scale, it assimilates ambient materials. Freshwater mussels have served as references for trophic studies of freshwater communities and as indicators of change in nutrient pollution load or source. Their suitability as reference animals has not yet been fully explored, however. We conducted a series of studies examining the suitability of freshwater mussels as isotopic baselines, using their ability to reflect variation in ambient nutrient loads as a case scenario. (1) We analyzed bivalve foot tissue delta(15)N and delta(13)C from 22 stream reaches in the Piedmont region of North Carolina, USA to show that compositions varied substantially among locations. Site mean bivalve delta(13)C values correlated with site ambient particulate organic matter (POM) delta(13)C values, and site mean bivalve delta(15)N values correlated with site ambient water dissolved delta(15)N-NO(3) values. (2) Similarity of results among sample types demonstrated that the minimally invasive hemolymph sample is a suitable substitute for foot tissue in delta(15)N analyses, and that small sample sizes generate means representative of a larger population. Both findings can help minimize the impact of sampling on imperiled freshwater mussel populations. (3) In a bivalve transplantation study we showed that hemolymph delta(15)N compositions responded to a shift in ambient dissolved delta(15)N-NO(3), although slowly. The tissue turnover time for bivalve hemolymph was 113 days. We conclude that bivalves serve best as biomonitors of chronic, rather than acute, fluctuations in stream nutrient loads, and provide initial evidence of their suitability as time-integrated isotopic baselines for community studies.