Differential preservation of endogenous human and microbial DNA in dental calculus and dentin

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007–0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003–70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute \u3e 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure

Development and implementation of new technologies in the steel industry

At each successive stage in the R&D cycle leading to the commercialization of a new technology, management in any industry must reevaluate prospects and make decisions accordingly. However, for the steel industry the difficulties of this kind of decision-making throughout the usually lengthy R&D cycle are heightened by the very nature of the industry. For example, the large scale of its processes and the high capital costs for plants for new processes mean that the progressive scale-ups during a given development may themselves cost millions. From Bethlehem Steel's experience the author cites one new product and two major process developments to illustrate technical and economic considerations that enter into decision-making as R&D moves step by step toward the commercialization of new technology.

Group A Streptococcal M1 Protein Provides Resistance against the Antimicrobial Activity of Histones

Histones are essential elements of chromatin structure and gene regulation in eukaryotes. An unexpected attribute of these nuclear proteins is their antimicrobial activity. A framework for histone release and function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps, a specialized cell death process in which DNA-based structures containing histones are extruded to ensnare and kill bacteria. Investigating the susceptibility of various Gram-positive pathogens to histones, we found high-level resistance by one leading human pathogen, group A Streptococcus (GAS). A screen of isogenic mutants revealed that the highly surface-expressed M1 protein, a classical GAS virulence factor, was required for high-level histone resistance. Biochemical and microscopic analyses revealed that the N-terminal domain of M1 protein binds and inactivates histones before they reach their cell wall target of action. This finding illustrates a new pathogenic function for this classic GAS virulence factor, and highlights a potential innate immune evasion strategy that may be employed by other bacterial pathogens

Femtosecond optical transfection of individual mammalian cells

Laser-mediated gene transfection into mammalian cells has recently emerged as a powerful alternative to more traditional transfection techniques. In particular, the use of a femtosecond-pulsed laser operating in the near-infrared (NIR) region has been proven to provide single-cell selectivity, localized delivery, low toxicity and consistent performance. This approach can easily be integrated with advanced multimodal live-cell microscopy and micromanipulation techniques. The efficiency of this technique depends on an understanding by the user of both biology and physics. Therefore, in this protocol we discuss the subtleties that apply to both fields, including sample preparation, alignment and calibration of laser optics and their integration into a microscopy platform. The entire protocol takes similar to 5 d to complete, from the initial setup of the femtosecond optical transfection system to the final stage of fluorescence imaging to assay for successful expression of the gene of interest

NOD1 and NOD2 Interact with the Phagosome Cargo in Mast Cells: A Detailed Morphological Evidence

Mast cells (MC) play a key role in triggering the inflammatory process and share some functions with professional phagocytes. It is not clear whether or not the phagocytic process in MC follows the same route and has the same meaning of that of professional phagocytes. Herein we analyze in detail the structure of the phagosome in rat peritoneal mast cells (RPMC). The ultrastructural analysis of the phagosome, containing either model particles or bacteria, reveals that these vacuoles are very tight, and in several areas, their membrane seems to have dissolved. RPMC express NOD1 and NOD2 proteins whose role is to recognize intracellular foreign components and induce the production of pro-inflammatory mediators. Following Escherichia coli ingestion, both these molecules are found on the phagosome membrane and on ingested pathogens, together with phagosome maturation markers. These findings suggest that in RPMC the ingested cargo can, through interruptions of the phagosome membrane, interact directly with NODs, which act as switches in the process of cytokine production

The phylogenetic profile of mast cells

Mast cells (MCs) are tissue-based immune cells that participate to both innate and adaptive immunities as well as to tissue-remodelling processes. Their evolutionary history appears as a fascinating process, whose outline we can only partly reconstruct according to current remnant evidence. MCs have been identified in all vertebrate classes, and a cell population with the overall characteristics of higher vertebrate MCs is identifiable even in the most evolutionarily advanced fish species. In invertebrates, cells related to vertebrate MCs have been recognized in ascidians, a class of urochordates which appeared approximately 500 million years ago. These comprise the granular hemocyte with intermediate characteristics of basophils and MCs and the \u201ctest cell\u201d (see below). Both types of cells contain histamine and heparin, and provide defensive functions. The test cell releases tryptase after stimulation with compound 48/80. A leukocyte ancestor operating in the context of a primitive local innate immunity probably represents the MC phylogenetic progenitor. This cell was likely involved in phagocytic and killing activity against pathogens and operated as a general inducer of inflammation. This early type of defensive cell possibly expressed concomitant tissue- reparative functions. With the advent of recombinase activating gene (RAG)-mediated adaptive immunity in the Cambrian era, some 550 million years ago, and the emergence of early vertebrates, MC progenitors differentiated towards a more complex cellular entity. Early MCs probably appeared in the last common ancestor we shared with hagfish, lamprey, and sharks about 450-500 million years ago