Structure and function of methanogenic microbial communities in soils from flooded rice and upland soybean fields from Sanjiang plain, NE China.

About 50 years ago, most of the natural wetlands in northeast China, the Sanjiang plain, were converted to either flooded rice fields or to upland soybean fields. After the conversion, natural wetland soils were either managed as artificial wetland or as drained upland resulting in soil microbial community changes. The purpose of our study was to understand how methanogenic microbial communities and their functions had changed in the two different soils upon conversion, and whether these communities now exhibit different resistance/resilience to drying and rewetting. Therefore, we determined function, abundance and composition of the methanogenic archaeal and bacterial communities in two soils reclaimed from a Carex wetland 25 years ago. We incubated the soils under anoxic conditions and measured the rates and pathways of CH4 production by analyzing concentration and ?13C of CH4 and acetate in the presence and absence of methyl fluoride, an inhibitor of aceticlastic methanogenesis. We also analyzed the abundance of bacterial and archaeal 16S rRNA genes, and of mcrA (coding for a subunit of the methyl coenzyme M reductase) using qPCR. The composition of the archaeal and bacterial 16S rRNA genes was determined by using MiSeq illumina sequencing. Our results showed clear differences in structure and function of methanogenic archaeal communities in rice field soil versus upland soil. Furthermore, in both soils composition of bacteria and archaea changed after artificial drying and became less diverse. The archaeal and bacterial signature species in the two soils were also different. However, functional changes were similar, with rates of CH4 production and contribution of aceticlastic methanogenesis decreasing upon drying and rewetting in both soils

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

Placental restriction increases adipose leptin gene expression and plasma leptin and alters their relationship to feeding activity in the young lamb

Low birth weight and catch-up growth predict increased adiposity in children and adults. This may be due in part to leptin resistance, as adults who were born small exhibit increased plasma leptin concentration relative to adiposity. Placental restriction (PR), a major cause of intrauterine growth restriction, reduces size at birth and increases feeding activity and adiposity by 6 wk in sheep. We hypothesized that PR would increase plasma leptin concentration and alter its relationship with feeding activity and adiposity in young lambs. Body size, plasma leptin, feeding activity, adiposity, leptin, and leptin receptor gene expression in adipose tissue were measured (12 control, 12 PR). PR reduced size at birth and increased adiposity. Plasma leptin concentration decreased with age, but to a lesser extent after PR and correlated positively with adiposity similarly in control and PR. PR increased plasma leptin concentration and perirenal adipose tissue leptin expression. Feeding activity correlated negatively with plasma leptin concentration in controls, but positively after PR. PR increases adipose tissue leptin expression and plasma leptin concentration, however, this increased abundance of peripheral leptin does not inhibit feeding activity (suckling event frequency), suggesting PR programs resistance to appetite and energy balance regulation by leptin, leading to early onset obesity.Miles J. De Blasio, Dominique Blache, Kathryn L. Gatford, Jeffrey S. Robinson and Julie A. Owen