Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.

No abstract

Abstract--The swelling of an n-butylammonium vermiculite in solutions of n-butylammonium chloride has been studied by neutron diffraction as a function of temperature and the concentration of the soaking solution. On heating a swollen sample a transition to the crystalline phase took place at a well-defined temperature, the c-axis spacing changing from 120 to 19.4 ,~ at 14~ in a 0.1 M solution and from 330 to 19.4 ~ at 33~ in a 0.01 M solution. The phase transition was completely reversible, and a study of the temperature-concentration phase diagram was made as high as 50~ The swollen phase was studied in a range of concentrations of the external solution between 0.2 M and 5 • 10 -4 M, for which the caxis spacings were 85 and 910 ~, respectively. The reversibility, sharpness, and reproducibility of the phase change from crystalline to swollen gel suggest that the transition is truly thermodynamic, which is not in accord with DLVO theory. The concentration dependence of the observed d-values also could not be explained satisfactorily by DLVO theory.

Components of cannabis, including THC, may decelerate peripheral monocyte processes that are implicated in HIV-associated neuroinflammation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent aryl hydrocarbon receptor agonist that elicits dose-dependent hepatic fat accumulation and inflammation that can progress to steatohepatitis. To investigate intestine-liver interactions that contribute to TCDD-elicited steatohepatitis, we examined the dose-dependent effects of TCDD (0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 µg/kg) on jejunal epithelial gene expression in C57BL/6 mice orally gavaged every 4 days for 28 days. Agilent 4x44K whole-genome microarray analysis of the jejunal epithelium identified 439 differentially expressed genes (|fold change| ≥ 1.5, P1(t) ≥ 0.999) across 1 or more doses, many related to lipid metabolism and immune system processes. TCDD-elicited differentially expressed genes were associated with lipolysis, fatty acid/cholesterol absorption and transport, the Kennedy pathway, and retinol metabolism, consistent with increased hepatic fat accumulation. Moreover, several major histocompatibility complex (MHC) class II genes (H2-Aa, H2-Ab1, H2-DMb1, Cd74) were repressed, coincident with decreased macrophage and dendritic cell levels in the lamina propria, suggesting migration of antigen-presenting cells out of the intestine. In contrast, hepatic RNA-Seq analysis identified increased expression of MHC class II genes, as well as chemokines and chemokine receptors involved in macrophage recruitment (Ccr1, Ccr5, Ccl5, Cx3cr1), consistent with hepatic F4/80 labeling and macrophage infiltration into the liver. Collectively, these results suggest TCDD elicits changes that support hepatic lipid accumulation, macrophage migration, and the progression of hepatic steatosis to steatohepatitis.