実験的に口腔StreptococcusおよびVeillonella種のバイオフィルムを産生させるワイヤー法(The wire method for generating experimental biofilms formed by oral Streptococcus and Veillonella species)

口腔細菌のバイオフィルムを効果的に形成させる方法の確立を目的として、歯列矯正に汎用されるワイヤーを足場とする培養装置を調製し、これを用いて口腔細菌を培養し、バイオフィルムの形成を調べた。ワイヤーは直径0.9mm、長さ15cmのものを用い、両端にゴム栓および軟質プラスチック菅で固定化し装置を調製し滅菌した。これをStreptococcusおよびVeillonella種の口腔細菌を培養した培地中に挿入し、37℃、4日間の嫌気培養をすると、ワイヤー上にバイオフィルムが形成されることが認められた。定量的実時間PCRを用いて、これらのバイオフィルムにおけるそれぞれの細菌を同定し、その細菌数を定量した。このワイヤー法はin vitro実験によるバイオフィルム産生に有用な方法であり、虫歯、歯周病および全身感染症のような口腔感染とバイオフィルム形成の関連性を調べるための有力な実験材料を提供するものである。口腔細菌のバイオフィルムを効果的に形成させる方法の確立を目的として、歯列矯正に汎用されるワイヤーを足場とする培養装置を調製し、これを用いて口腔細菌を培養し、バイオフィルムの形成を調べた。ワイヤーは直径0.9mm、長さ15cmのものを用い、両端にゴム栓および軟質プラスチック菅で固定化し装置を調製し滅菌した。これをStreptococcusおよびVeillonella種の口腔細菌を培養した培地中に挿入し、37℃、4日間の嫌気培養をすると、ワイヤー上にバイオフィルムが形成されることが認められた。定量的実時間PCRを用いて、これらのバイオフィルムにおけるそれぞれの細菌を同定し、その細菌数を定量した。このワイヤー法はin vitro実験によるバイオフィルム産生に有用な方法であり、虫歯、歯周病および全身感染症のような口腔感染とバイオフィルム形成の関連性を調べるための有力な実験材料を提供するものである

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Comparative Pan-Genome Analysis of Oral Veillonella Species

The genus Veillonella is a common and abundant member of the oral microbiome. It includes eight species, V. atypica, V. denticariosi, V. dispar, V. infantium, V. nakazawae, V. parvula, V. rogosae and V. tobetusensis. They possess important metabolic pathways that utilize lactate as an energy source. However, the overall metabolome of these species has not been studied. To further understand the metabolic framework of Veillonella in the human oral microbiome, we conducted a comparative pan-genome analysis of the eight species of oral Veillonella. Analysis of the oral Veillonella pan-genome revealed features based on KEGG pathway information to adapt to the oral environment. We found that the fructose metabolic pathway was conserved in all oral Veillonella species, and oral Veillonella have conserved pathways that utilize carbohydrates other than lactate as an energy source. This discovery may help to better understand the metabolic network among oral microbiomes and will provide guidance for the design of future in silico and in vitro studies