Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum

Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.     

Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy

Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.     

The oxygen resources of the hypolimnion of ionically enriched onondaga lake,NY, U.S.A.

The depletion of hypolimnetic DO and the upper depth boundary of anoxia for four different years (1978, 1979, 1980 and 1981), and the accumulation of sulfide for a single year (1981), are documented for ionically enriched hypereutrophic Onondaga Lake, NY, USA. The depletion rate, represented as the areal hypolimnetic oxygen deficit (AHOD, g m^?2 day^?1), was extremely high (1.2–2.7 g m^?2 day^?1), The large differences in the rate within individual years and from year to year were largely a result of differences in attendant vertical mixing (parameterized as the hypolimnetic heating rate). The entire hypolimnion (depth interval from 11 to 20 m) was without O₂ by late June of all 4 yr; anoxia was observed above the hypolimnion on some occasions when secondary stratification occurred. Sulfide accumulated progressively in the hypolimnion in 1981 following the onset of anoxia to a volume weighted concentration of I1 mg L^?1. The ionic discharge from an adjoining alkali manufacturer exacerbated the problem of limited O₂ resources of the hypolimnion by: (1) decreasing vertical mixing, (2) prolonging the duration of stratification, (3) causing abbreviated turnovers, and (4) encouraging increased rates of phytoplankton settling.     

At-sea distribution of waved albatrosses and the Galápagos Marine Reserve

Albatross populations worldwide are threatened by incidental takes in longline fishery operations. The recent establishment of the Galápagos Marine Reserve (GMR) is relevant to the longline bycatch issue, as it prohibits industrial longlining in the vicinity of the major nesting site of waved albatrosses (Phoebastria irrorata). However, the legality of the fishing protections is being challenged, highlighting a need for data on use of the GMR by albatrosses. We used satellite tracking over a total of four breeding seasons to determine the distribution of waved albatrosses inside and outside the GMR, and thereby assess the degree of protection that GMR provisions offer to this species. During the incubation period, breeding adults made commuting trips from the nesting island (Isla Española) to the Peruvian upwelling zone, traveling north, east, and south after leaving the nest. During the brooding period, the distribution contracted markedly, and most satellite fixes were within the GMR. During the rearing period, breeders performed both long trips outside the GMR and short trips within. The southeastern portion of the GMR is used throughout the incubation, brooding, and early rearing periods by breeding waved albatrosses. Indirect information from non-breeding adults indicates that they are likely to use the waters of the GMR extensively.     

Evaluation of a radioimmunoassay for type III procollagen peptide in the dog

Type III procollagen peptide (P-3-P) is a serum marker for hepatic fibrosis in humans. The utility of a commercially available radioimmunoassay for P-3-P was evaluated in the dog. The specificity of the assay was assessed by polyacrylamide gel electrophoresis (PAGE) of canine serum and purified bovine P-3-P, followed by Western immunoblotting with rabbit aniti-P-3-P serum. The sensitivity was assessed by performing the radioimmunoassay on dilutions of sera from 22 dogs. Polyacrylamide gel electrophoresis of purified bovine P-3-P and sera from two dogs suspected of having elevated P-3-P concentrations revealed no homologous bands of staining. Western immunoblotting showed marked cross-reactivity of the high antisera concentrations with several components of the serum proteins, but none corresponding to the purified P-3-P. All tested sera from dogs had minimal competitive binding with radiolabeled P-3-P in the radioimmunoassay. Dilution curves of dog sera did not parallel either the standard curve or the dilution curve of a known test human serum. There were no statistically different P-3-P concentrations in any of the groups of dogs studied. It was concluded that currently available radioimmunoassay kits for the measurement of P-3-P in the human are not applicable in the dog. Seemingly, the structure or metabolism of canine P-3-P may vary significantly from that of the bovine or human, limiting the sensitivity and specificity of this assay in the dog.     

The susceptibility of bovine udder quarters colonized with Corynebacterium bovis to experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

Twenty bovine udder quarters colonized with Corynebacterium bovis SR6 and 20 uncolonized quarters were challenged by inoculation of Staphylococcus aureus Newbould 305 (ATCC 29740) into the teat cistern. The percentage of infection in quarters colonized with C. bovis (50%) was significantly lower than that in controls (100%). By similar challenge no significant difference was observed between the percentage of infection with Streptococcus agalactiae ATCC 27956 in 33 quarters colonized with C. bovis (70%) compared to 33 controls (87.9%). A total of 37 quarters colonized with C. bovis and 37 control quarters were challenged with Staph. aureus Newbould 305 (ATCC 29740) and Maxi (ATCC 27543) and Strep. agalactiae (ATCC 27956) by exposure of the teat orifice. The percentage of teat ducts colonized with C. bovis which became infected with either pathogen was not different from that for controls.