Resistance, resilience and recovery: aquatic bacterial dynamics after water column disturbance

For lake microbes, water column mixing acts as a disturbance because it homogenizes thermal and chemical gradients known to define the distributions of microbial taxa. Our first objective was to isolate hypothesized drivers of lake bacterial response to water column mixing. To accomplish this, we designed an enclosure experiment with three treatments to independently test key biogeochemical changes induced by mixing: oxygen addition to the hypolimnion, nutrient addition to the epilimnion, and full water column mixing. We used molecular fingerprinting to observe bacterial community dynamics in the treatment and control enclosures, and in ambient lake water. We found that oxygen and nutrient amendments simulated the physical-chemical water column environment following mixing and resulted in similar bacterial communities to the mixing treatment, affirming that these were important drivers of community change. These results demonstrate that specific environmental changes can replicate broad disturbance effects on microbial communities. Our second objective was to characterize bacterial community stability by quantifying community resistance, recovery and resilience to an episodic disturbance. The communities in the nutrient and oxygen amendments changed quickly (had low resistance), but generally matched the control composition by the 10th day after treatment, exhibiting resilience. These results imply that aquatic bacterial assemblages are generally stable in the face of disturbance.     

Design considerations for PAMAM dendrimer therapeutics

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer–ester MTX conjugates were much more cytotoxic than the dendrimer–amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics.     

Persistent Popular Images of Pastoralists

Pastoralists have long been recurrent figures in visual images and other representations of Africa and Africans. Whether seen in positive or negative terms, pastoralists have provided means for thinking about and imaging cultural difference and identity, with considerable continuity in representational forms and themes. As popular visual media proliferated and changed over the past two centuries--from postcards, trade cards, and live shows to Hollywood films and video games--African pastoralists have continued to appear in each new form, often replicating the types and stereotypes of Euroamerican understandings even as they register new and varied circumstances. The proliferation and reverberation of similar images through diverse visual media is one way these images have come to seem "natural" and to develop such remarkable persistence [Kratz 2002]. Using cases drawn from eastern and southern Africa, this collection of articles considers the multifaceted processes of representation involved in imaging African pastoralists. It invites attention to how such representations are produced in diverse visual media and through interconnections among visual and verbal media, examining the range of actors, interactions, and mediations involved in crafting representations of African pastoralists at different times and in different places.     

Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells

Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many—but not all—herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this, we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation, or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a postendocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless.     

Measurement of DNA damage induced by irradiation with gamma-rays from a TRIGA Mark II research reactor in human cells using Fast Micromethod.

The Fast Micromethod is a novel quick and convenient microplate assay for determination of DNA single-strand breaks. This method measures the rate of unwinding of cellular DNA upon exposure to alkaline conditions using a fluorescent dye which preferentially binds to double-stranded DNA. Here we applied this method to determine the levels of DNA single-strand breaks in HeLa cells induced by y-irradiation deriving from fission isotopes and activation products at the TRIGA Mark II research reactor in Mainz. An increased strand scission factor (SSF) value, which is indicative for DNA damage, was found at doses of 1 Gy and higher. A similar increase in SSF value, which further increased in a dose-dependent manner, was found in human peripheral blood mononuclear cells after irradiation with 6 MV X-rays from a linear accelerator to give a total exposure of 0.5 to 10 Gy.     

Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IkappaB kinase promotes phosphorylation and degradation of NFkappaB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IkappaB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.     

Hypomorphic temperature-sensitive alleles of NSDHL cause CK syndrome

CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development.     

Expression of the ATP-binding cassette transporter gene ABCG1 (ABC8) in Tangier disease

Several members of the ATP-binding cassette (ABC) transporter family are involved in cholesterol efflux from cells. A defect in one member, ABCA1, results in Tangier disease, a condition characterized by cholesterol accumulation in macrophages and virtual absence of mature circulating high-density lipoproteins. Expression of a second member, ABCG1, is increased by cholesterol-loading in human macrophages. We now show that ABCG1, which we identified by differential display RT-PCR in foamy macrophages, is overexpressed in macrophages from patients with Tangier disease compared to control macrophages. On examination by confocal laser scanning microscopy, ABCG1 was present in perinuclear structures within the cell. In addition, a combination of in situ hybridization and indirect immunofluorescence microscopy revealed that ABCG1 is expressed in foamy macrophages within the atherosclerotic plaque. These data indicate that not only ABCA1 but also ABCG1 may play a role in the cholesterol metabolism of macrophages in vitro and in the atherosclerotic plaque.