Analysis of individual cells identifies cell‐to‐cell variability following induction of cellular senescence

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.     

Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

Stable isotopes as indicators of wastewater effects on the macroinvertebrates of urban rivers

Rivers in urban locations frequently receive contaminated wastewater and particulate waste either directly from storm overflows or from sewage treatment facilities. Although many urban streams are now recovering from wide-scale historic pollution, lower-level effects on water chemistry, nutrients and biotic composition are still widespread. We aimed to determine whether such effects could be detected using stable isotope ratios (??¹⁵N, ??¹³C and ??³⁴S) in macroinvertebrates alone or in conjunction with traditional biomonitoring. Macroinvertebrates were collected upstream and downstream of 11 different secondary wastewater treatment works (WwTW) in South Wales and the Welsh borders (United Kingdom). Overall, mean invertebrate ??¹⁵N signatures downstream of the WwTW were significantly enriched despite variation amongst sites. Moreover, changes between upstream and downstream macroinvertebrate ??¹⁵N values were highly correlated with patterns in macroinvertebrate community composition, increased total macroinvertebrate abundance, and reduced Shannon Diversity and other biomonitoring indices (% EPT, % shredders and ASPT scores). Changes in invertebrate ??¹⁵N values also paralleled the consented discharge volumes and population equivalents from each WwTW. In contrast, isotopic ratios of ??¹³C and ??³⁴S were unable to distinguish or quantify wastewater input into the rivers but differences were apparent amongst study streams. Overall, these results suggest that macroinvertebrate ??¹⁵N signatures can detect and quantify the effects of secondary sewage treatment inputs to riverine ecosystems. Moreover, the method potentially provides a sensitive means for tracing sewage-derived nutrients into food webs while inferring effects on aquatic communities where sewage-loads are subtle or confounded by other stressors.     

Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked.

Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).     

Sedimentological evidence for rooting structures in the Early Devonian Anglo-Welsh Basin (UK), with speculation on their producers

Rooting structures preserved as casts in alluvial deposits of the Lower Devonian Lower Old Red Sandstone occur more extensively than previously thought in the Anglo-Welsh Basin. Two rooting structure morphotypes are identified: morphotype 1 being predominantly horizontal, and morphotype 2 comprising vertical forms. Both morphotypes taper along their length, are oval in cross-section, branch, and can be linear or sinuous. Fills are heterolithic in nature. The rooting structures are observed in sediments deposited in a wide variety of environments including both low-sinuosity within-channel bar-tops and accretionary bar surfaces (inclined-heterolithic stratification); accretionary banks of sinuous ephemeral channels (inclined-heterolithic stratification); floodplains (including margins of shallow floodplain ponds) of both ephemeral and perennial river channels, and well-developed calcic Vertisols. Although a vascular plant origin to the rooting structures cannot be discounted, there is circumstantial evidence that the structures were produced by the enigmatic Prototaxites, recently reinterpreted as a giant fungus. It is possible that they represent underground aggregates (rhizomorphs or cords) of hyphae involved in anchorage and nutrient foraging. Organic matter associated with biofilms and crusts is hypothesized as a source of nutrients for the presumed saprotroph.     

Response of the Photosynthetic Apparatus in Dunaliella salina (Green Algae) to Irradiance Stress

The response of the photosynthetic apparatus in the green alga Dunaliella salina, to irradiance stress was investigated. Cells were grown under physiological conditions at 500 millimoles per square meter per second (control) and under irradiance-stress conditions at 1700 millimoles per square meter per second incident intensity (high light, HL). In control cells, the light-harvesting antenna of photosystem I (PSI) contained 210 chlorophyll a/b molecules. It was reduced to 105 chlorophyll a/b in HL-grown cells. In control cells, the dominant form of photosystem II (PSII) was PSII_α(about 63% of the total PSII) containing >250 chlorophyll a/b molecules. The smaller antenna size PSII_β centers (about 37% of PSII) contained 135 ± 10 chlorophyll a/b molecules. In sharp contrast, the dominant form of PSII in HL-grown cells accounted for about 95% of all PSII centers and had an antenna size of only about 60 chlorophyll a molecules. This newly identified PSII unit is termed PSII_γ. The HL-grown cells showed a substantially elevated PSII/PSI stoichiometry ratio in their thylakoid membranes (PSII/PSI = 3.0/1.0) compared to that of control cells (PSII/PSI = 1.4/1.0). The steady state irradiance stress created a chronic photoinhibition condition in which D. salina thylakoids accumulate an excess of photochemically inactive PSII units. These PSII units contain both the reaction center proteins and the core chlorophyll-protein antenna complex but cannot perform a photochemical charge separation. The results are discussed in terms of regulatory mechanism(s) in the plant cell whose function is to alleviate the adverse effect of irradiance stress.     

The influence of IL-1 treatment on the reconstitution of the hemopoietic and immune systems after sublethal radiation

The influence of IL-1 administration on the recovery of the hemopoietic and immune systems from sublethal irradiation was assessed. Mice were irradiated (750 R) and injected twice daily with purified recombinant derived IL-1 beta (200 ng/injection). At various times after irradiation, the functional capacity of the hemopoietic and immune systems was determined. It was found that IL-1 therapy resulted in a significantly greater number of granulocyte-macrophage-CSF responsive colony-forming cells in the bone marrow of the irradiated mice on days 5 and 11 postirradiation but not at later times. In addition the radiation induced neutropenia recovered quicker in the IL-1-treated mice with significantly greater numbers of peripheral blood granulocytes being seen on days 15 and 20 after irradiation. The influence of IL-1 therapy on the recovery of the immune system was also assessed. Of note was the observation that mice receiving IL-1 therapy had chronically hypoplastic thymi. Although thymic cellularity increased with time after irradiation in the control mice, there was no such increase in the IL-1-treated mice. Similarly, the number of pre-B cells in the marrow of these mice was also diminished. Thus, in the IL-1-treated mice the regeneration of the peripheral immune function was retarded, characterized by a general lymphopenia and decreased splenic responses to mitogenic stimuli.     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.