A Type 2C Protein Phosphatase FgPtc3 Is Involved in Cell Wall Integrity, Lipid Metabolism, and Virulence in Fusarium graminearum

Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum

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

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Spectral response analysis: An indirect and non-destructive methodology for the chlorophyll quantification of biocrusts

Chlorophyll a concentration (Chla) is a well-proven proxy of biocrust development, photosynthetic organisms' status, and recovery monitoring after environmental disturbances. However, laboratory methods for the analysis of chlorophyll require destructive sampling and are expensive and time consuming. Indirect estimation of chlorophyll a by means of soil surface reflectance analysis has been demonstrated to be an accurate, cheap, and quick alternative for chlorophyll retrieval information, especially in plants. However, its application to biocrusts has yet to be harnessed. In this study we evaluated the potential of soil surface reflectance measurements for non-destructive Chla quantification over a range of biocrust types and soils. Our results revealed that fromthe different spectral transformation methods and techniques, the first derivative of the reflectance and the continuum removal were the most accurate for Chla retrieval. Normalized difference values in the red-edge region and common broadband indexes (e.g., normalized difference vegetation index (NDVI)) were also sensitive to changes in Chla. However, such approaches should be carefully adapted to each specific biocrust type. On the other hand, the combination of spectral measurements with non-linear random forest (RF) models provided very good fits (R > 0.94) with a mean root mean square error (RMSE) of about 6.5 μg/g soil, and alleviated the need for a specific calibration for each crust type, opening a wide range of opportunities to advance our knowledge of biocrust responses to ongoing global change and degradation processes from anthropogenic disturbance.This research was funded by the RESUCI (CGL2014-59946-R), DINCOS (CGL2016-78075-P), and REBIOARID (RTI2018-101921-B-I00) projects funded by the Spanish National Plan for Research and the European Union ERDF funds, and the project H2020-MSCA-RISE-GYPWORLD (funded by the European Union’s Horizon 2020 research and innovation program under the Marie Slodowska-Curie Grant Agreement No. 777803). Field sampling was conducted within the framework of the project “Cuantificación de flujos de carbono y agua en zonas áridas a partir de información spectral” founded by Aerial Platforms for Research-ICTS (INTA) throughout the campaign “Investigaciones de Altura.” J.R.R. was funded by the FPU predoctoral fellowship from the Educational, Culture and Sports Ministry of Spain (FPU14/05806). E.R.-C. was supported by the Juan de la Cierva incorporación fellowship (IJCI-2016-29274) and by the Hipatia postdoctoral fellowship funded by the University of Almería. B.R.-R. was supported by the foundation Tatiana Pérez de Guzmán el Bueno under its predoctoral fellowship program and S.C. was supported by the Hipatia postdoctoral fellowship funded by the University of Almería

Spectral response analysis: An indirect and non-destructive methodology for the chlorophyll quantification of biocrusts

Chlorophyll a concentration (Chla) is a well-proven proxy of biocrust development, photosynthetic organisms' status, and recovery monitoring after environmental disturbances. However, laboratory methods for the analysis of chlorophyll require destructive sampling and are expensive and time consuming. Indirect estimation of chlorophyll a by means of soil surface reflectance analysis has been demonstrated to be an accurate, cheap, and quick alternative for chlorophyll retrieval information, especially in plants. However, its application to biocrusts has yet to be harnessed. In this study we evaluated the potential of soil surface reflectance measurements for non-destructive Chla quantification over a range of biocrust types and soils. Our results revealed that fromthe different spectral transformation methods and techniques, the first derivative of the reflectance and the continuum removal were the most accurate for Chla retrieval. Normalized difference values in the red-edge region and common broadband indexes (e.g., normalized difference vegetation index (NDVI)) were also sensitive to changes in Chla. However, such approaches should be carefully adapted to each specific biocrust type. On the other hand, the combination of spectral measurements with non-linear random forest (RF) models provided very good fits (R > 0.94) with a mean root mean square error (RMSE) of about 6.5 μg/g soil, and alleviated the need for a specific calibration for each crust type, opening a wide range of opportunities to advance our knowledge of biocrust responses to ongoing global change and degradation processes from anthropogenic disturbance.This research was funded by the RESUCI (CGL2014-59946-R), DINCOS (CGL2016-78075-P), and REBIOARID (RTI2018-101921-B-I00) projects funded by the Spanish National Plan for Research and the European Union ERDF funds, and the project H2020-MSCA-RISE-GYPWORLD (funded by the European Union’s Horizon 2020 research and innovation program under the Marie Slodowska-Curie Grant Agreement No. 777803). Field sampling was conducted within the framework of the project “Cuantificación de flujos de carbono y agua en zonas áridas a partir de información spectral” founded by Aerial Platforms for Research-ICTS (INTA) throughout the campaign “Investigaciones de Altura.” J.R.R. was funded by the FPU predoctoral fellowship from the Educational, Culture and Sports Ministry of Spain (FPU14/05806). E.R.-C. was supported by the Juan de la Cierva incorporación fellowship (IJCI-2016-29274) and by the Hipatia postdoctoral fellowship funded by the University of Almería. B.R.-R. was supported by the foundation Tatiana Pérez de Guzmán el Bueno under its predoctoral fellowship program and S.C. was supported by the Hipatia postdoctoral fellowship funded by the University of Almería

Assessing the influence of soil abiotic and biotic factors on Nostoc commune inoculation success

Aims: A possible approach to restore drylands is to recover biocrusts by inoculating cyanobacteria. Many studies have demonstrated the ability of cyanobacteria to successfully colonize soil and improve its functions. However, most studies have focused on the abiotic factors influencing the inoculation success, overlooking biotic factors. We examined the influence of the soil indigenous community on the inoculated cyanobacteria by sterilizing soils and analyzing its effects on several key soil properties. Methodology: Nostoc commune was inoculated under laboratory conditions on two soil types with different levels of degradation and under two watering frequencies. All treatments were carried out in natural and sterilized soils. Results: The analysis of the cyanobacterial coverage (30–50% in all inoculated soils), chlorophyll a, visible albedo, roughness, water repellency, organic carbon (OC) and exopolysaccharides (EPS) content showed that cyanobacterial inoculation succeeded, improving soil properties that varied depending on the soil type. The sterilization of soils had little effect: in less degraded soils, the results suggest a faster growth of the indigenous community reaching coverage values of 10% and an interaction with the inoculum that led to a decrease in 2 and 0.3 g Kg of OC and TB-EPS, respectively; in more degraded soils, the community reduced the cyanobacterial coverage around 11%, suggesting competitive interactions. Conclusions: N. commune can be employed to restore lifeless soils. Also, the analysis of native soil community should be considered before field inoculation to plan appropriate methodologies

Land degradation effects on composition of pioneering soil communities: An alternative successional sequence for dryland cyanobacterial biocrusts

In drylands, soil surfaces in interplant spaces are usually covered by biocrusts, which consist of communities of heterotrophic and chemoautotrophic bacteria, cyanobacteria, mosses, lichens, microalgae, fungi and other organisms. Cyanobacteria are of special interest because of their capacity to promote biocrust succession or increase soil fertility and stability. Therefore, some studies have analyzed their communities in different ecosystems, focusing on how different factors, such as temperature or altitude, influence their composition. Nevertheless, to our knowledge, the relationship between ecosystem degradation and cyanobacterial community composition has not yet been studied in depth. This could be determinant for the successful development of tools for restoring degraded biocrusts by cyanobacterial inoculation. Thus, the objective of this study was to analyze the effect of the ecosystem degradation level on cyanobacteria composition from topsoil communities, where they are keystone pioneering organisms. To do this, we analyzed the cyanobacterial diversity by molecular sequencing (16S rRNA gene) of the DNA extracted from biocrusts at different developmental stages, which were collected from three ecosystems in southeastern Spain. The selected ecosystems represent different “land-condition” states as a result of degradation processes. In one of them soil was removed by mining (the Gador quarry), the second is a natural badland area (El Cautivo) where water erosion is intense, and the third ecosystem is a well-preserved area (Balsa Blanca). Our findings show that cyanobacterial richness decreases (up to 28 OTUs) as degradation increases and biocrust developmental stage decreases. Also, the relative abundances of most of the species were significantly correlated with the degradation state of the sampling site, either showing a positive or negative trend. Two of the species which increased in abundance with site degradation, and were especially abundant in incipient biocrusts, were Leptolyngbya frigida and Trichocoleus desertorum, while other species, also showing an increase in abundance with degradation, but having a higher relative abundance in most developed biocrusts, were Nostoc commune, Tolypothrix distorta and Scytonema sp. The significant correlation of these species with degradation at different biocrusts developmental stages, suggests an alternative developmental sequence for drylands, at least in more degraded ecosystems. In less degraded ones, the composition of the major cyanobacterial groups followed the common pattern of bundle-forming cyanobacteria (54.7%) pertaining to the Microcoleus genus followed by other non-heterocystous filamentous (17.4%), unicellular/colonial (7.5%) and heterocystous cyanobacteria (1.1%). In comparison, the cyanobacterial groups dominating the most incipient biocrusts, colonizing the most degraded soil, were the filamentous non-heterocystous (50.7%) and the bundleforming cyanobacteria (48.9%). Therefore, our results show that some cyanobacterial species, which do not belong to traditional pioneer genera, are frequent colonizers of degraded soils, and then, they could be potentially used for producing a more efficient inoculum for inducing biocrust formation and restoring degraded soils. Finally, it is also remarkable that L. frigida appears as dominant in some biocrusts from drylands (up to 74.9% of abundance) being therefore demonstrated its wide distribution in nonpolar biomes and its capacity to also inhabit degraded arid soilsThis work was supported by the projects RESUCI (CGL 2014-59946-R), the project REBIOARID (RTI 2018-101921-B-I00), and the GL 2013-44870-R and GL 2017-6258-R projects, all of them founded by the Spanish National Plan for Research and the European Union ERDF fund

Effects of dietary bread crust Maillard reaction products on calcium and bone metabolism in rats

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Cyanobacteria inoculation enhances carbon sequestration in soil substrates used in dryland restoration

Despite significant efforts to restore dryland ecosystems worldwide, the rate of success of restoration is extremely low in these areas. The role of cyanobacteria from soil biocrusts in reestablishing soil functions of degraded land has been highlighted in recent years. These organisms are capable of improving soil structure and promoting soil N and C fixation. Nevertheless, their application to restore functions of reconstructed soils in dryland restoration programs is yet to be harnessed. In this study, we used microcosms under laboratory conditions to analyse the effects of inoculating soil substrates used in post-mine restoration with a mixture of N-fixing cyanobacteria isolated from soil biocrust (Nostoc commune, Tolypothrix distorta and Scytonema hyalinum) on i) the recovery of the biocrust, and ii) the carbon sequestration and mineralisation rates of these substrates. Soils were collected from an active mine site in the mining-intensive biodiverse Pilbara region (north-west Western Australia) and consisted of previously stockpiled topsoil, overburden waste material, a mixture of both substrates, and a natural soil from an undisturbed area. Our results showed that cyanobacteria rapidly colonised the mine substrates, with biocrust coverage ranging from 23.8 to 52.2% and chlorophyll a concentrations of up to 12.2 μg g−1 three months after inoculation. Notably, soil organic C contents increased 3-fold (P < 0.001) in the mine waste substrate (from 0.6 g kg−1 to 1.9 g kg−1) during this period of time. Overall, our results showed that cyanobacteria inoculation can rapidly modify properties of reconstructed soil substrates, underpinning the potential key role of these organisms as bio-tools to initiate recovery of soil functions in infertile, reconstructed soil substrates