Publisher Summary This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

Sequencing end-labeled DNA with base-specific chemical cleavages.

Neutrophils have traditionally been thought of as simple foot soldiers of the innate immune system with a restricted set of pro-inflammatory functions. More recently, it has become apparent that neutrophils are, in fact, complex cells capable of a vast array of specialized functions. Although neutrophils are undoubtedly major effectors of acute inflammation, several lines of evidence indicate that they also contribute to chronic inflammatory conditions and adaptive immune responses. Here, we discuss the key features of the life of a neutrophil, from its release from bone marrow to its death. We discuss the possible existence of different neutrophil subsets and their putative anti-inflammatory roles. We focus on how neutrophils are recruited to infected or injured tissues and describe differences in neutrophil recruitment between different tissues. Finally, we explain the mechanisms that are used by neutrophils to promote protective or pathological immune responses at different sites.

Neutrophil recruitment and function in health and inflammation

Viroids are uncoated infectious RNA molecules pathogenic to certain higher plants. Four different highly purified viroids were studied. By ultracentrifugation, thermal denaturation, electron microscopy, and end group analysis the following features were established: (i) the molecular weight of cucumber pale fruit viroid from tomato is 110,000, of citrus exocortis viroid from Gynura 119,000, of citrus exocortis viroid from tomato 119,000 and of potato spindle tuber viroid from tomato 127,000. (ii) Viroids are single-stranded molecules. (iii) Virods exhibit high thermal stability, cooperativity, and self-complementarity resulting in a rod-like native structure. (iv) Viroids are covalently closed circular RNA molecules.

https://www.pnas.org/content/pnas/73/11/3852.full.pdf

Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures.

A dot hybridization method is presented for rapidly determining the relative concentrations of nucleic acids in a mixture, as well as the extent of sequence homology between related RNA or DNA species.

Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure

'If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo' (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future.

G-quadruplexes and their regulatory roles in biology.

Here we investigate the dynamics of the hepatic intravascular immune response to a pathogen relevant to invariant natural killer T cells (iNKT cells). Immobilized Kupffer cells with highly ramified extended processes into multiple sinusoids could effectively capture blood-borne, disseminating Borrelia burgdorferi, creating a highly efficient surveillance and filtering system. After ingesting B. burgdorferi, Kupffer cells induced chemokine receptor CXCR3-dependent clustering of iNKT cells. Kupffer cells and iNKT cells formed stable contacts via the antigen-presenting molecule CD1d, which led to iNKT cell activation. An absence of iNKT cells caused B. burgdorferi to leave the blood and enter the joints more effectively. B. burgdorferi that escaped Kupffer cells entered the liver parenchyma and survived despite Ito cell responses. Kupffer cell-iNKT cell interactions induced a key intravascular immune response that diminished the dissemination of B. burgdorferi.

/pdf/an-intravascular-immune-response-to-borrelia-burgdorferi-55jm20zrld.pdf

An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells

Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood–brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.

http://www.murakamicentreforlyme.org/Documents/High%20Res%203D%20spirochete.pdf

Real-time high resolution 3D imaging of the lyme disease spirochete adhering to and escaping from the vasculature of a living host.

The linear plasmid, lp28-1, is required for persistent infection by the Lyme disease spirochete, Borrelia burgdorferi. This plasmid contains the vls antigenic variation locus, which has long been thought to be important for immune evasion. However, the role of the vls locus as a virulence factor during mammalian infection has not been clearly defined. We report the successful removal of the vls locus through telomere resolvase-mediated targeted deletion, and demonstrate the absolute requirement of this lp28-1 component for persistence in the mouse host. Moreover, successful infection of C3H/HeN mice with an lp28-1 plasmid in which the left portion was deleted excludes participation of other lp28-1 non-vls genes in spirochete virulence, persistence and the process of recombinational switching at vlsE. Data are also presented that cast doubt on an immune evasion mechanism whereby VlsE directly masks other surface antigens similar to what has been observed for several other pathogens that undergo recombinational antigenic variation.

The role of VlsE antigenic variation in the Lyme disease spirochete: persistence through a mechanism that differs from other pathogens

A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.

End labeling of enzymatically decapped mRNA

Hematogenous dissemination is important for infection by many bacterial pathogens, but is poorly understood because of the inability to directly observe this process in living hosts at the single cell level. All disseminating pathogens must tether to the host endothelium despite significant shear forces caused by blood flow. However, the molecules that mediate tethering interactions have not been identified for any bacterial pathogen except E. coli, which tethers to host cells via a specialized pillus structure that is not found in many pathogens. Furthermore, the mechanisms underlying tethering have never been examined in living hosts. We recently engineered a fluorescent strain of Borrelia burgdorferi, the Lyme disease pathogen, and visualized its dissemination from the microvasculature of living mice using intravital microscopy. We found that dissemination was a multistage process that included tethering, dragging, stationary adhesion and extravasation. In the study described here, we used quantitative real-time intravital microscopy to investigate the mechanistic features of the vascular interaction stage of B. burgdorferi dissemination. We found that tethering and dragging interactions were mechanistically distinct from stationary adhesion, and constituted the rate-limiting initiation step of microvascular interactions. Surprisingly, initiation was mediated by host Fn and GAGs, and the Fn- and GAG-interacting B. burgdorferi protein BBK32. Initiation was also strongly inhibited by the low molecular weight clinical heparin dalteparin. These findings indicate that the initiation of spirochete microvascular interactions is dependent on host ligands known to interact in vitro with numerous other bacterial pathogens. This conclusion raises the intriguing possibility that fibronectin and GAG interactions might be a general feature of hematogenous dissemination by other pathogens.

/pdf/molecular-mechanisms-involved-in-vascular-interactions-of-1hbfe47d6h.pdf

George Chaconas

Papers

An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells

Real-time high resolution 3D imaging of the lyme disease spirochete adhering to and escaping from the vasculature of a living host.

The role of VlsE antigenic variation in the Lyme disease spirochete: persistence through a mechanism that differs from other pathogens

End labeling of enzymatically decapped mRNA

Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host.