Background: Microalbuminuria is an early sign of kidney disease in diabetes and indicates cardiovascular risk. We tested if a prespecified urinary proteomic risk classifier (CKD273) was associated with development of microalbuminuria and if progression to microalbuminuria could be prevented with the mineralocorticoid receptor antagonist spironolactone. Methods: Prospective multicentre study in people with type 2 diabetes, normal urinary albumin excretion and preserved renal function in 15 European specialist centres. High-risk individuals determined by CKD273 were randomised 1:1 (interactive web response system) in a double-blind randomised controlled trial comparing spironolactone 25 mg o.d. to placebo. Primary endpoint was development of confirmed microalbuminuria in all individuals with available data. Secondary endpoints included reduction in incidence of microalbuminuria with spironolactone and association between CKD273 and impaired renal function defined as a glomerular filtration rate < 60 ml/min per 1•73 m 2. This study is registered with ClinicalTrials.gov: NCT02040441 and is completed. Findings: From March 25, 2014 to September 30, 2018 we followed 1775 participants, 12% (n=216) had high-risk urinary proteomic pattern of which 209 were included in the trial and assigned spironolactone (n=102) or placebo (n=107). Median follow-up time was 2•51 years (IQR 2•0-3•0). Progression to microalbuminuria was seen in 28•2% of high-risk and 8•9% of low-risk people (P< 0•001) (hazard ratio (HR), 2•48; 95% confidence interval [CI], 1•80 to 3•42 P<0•001, independent of baseline clinical characteristics). A 30% decline in eGFR from baseline was seen in 42 (19•4 %) high-risk participants compared to 62 (3•9 %) low-risk participants, HR 5•15; 95 % CI (3•41 to 7•76; p<0.0001). Development of microalbuminuria was seen in 35 (33%) randomised to placebo and 26 (25%) randomised to spironolactone treatment (HR 0•81, 95% CI, 0•49 to 1•34, P=0•41). Harms: hyperkalaemia was seen in 13 versus 4, and gynaecomastia in 3 versus 0 subjects on spironolactone and placebo, respectively. Interpretation: In people with type 2 diabetes and normoalbuminuria, the urinary proteomic classifier CKD273 was associated with a 2•5 times increased risk for progression to microalbuminuria over a median of 2•5 years, independent of clinical characteristics. Spironolactone did not prevent progression to microalbuminuria in high-risk subjects.

The SWI/SNF complex is a key element of the yeast CWI MAPK pathway, which mediates the chromatin remodeling necessary for an adequate transcriptional response to cell wall stress. The MAPK Slt2 mediates, through Rlm1, nucleosome rearrangements at cell wall stress–responsive genes by targeting the SWI/SNF complex.

Fungi are surrounded by an essential structure, the cell wall, which not only confers cell shape but also protects cells from environmental stress. As a consequence, yeast cells growing under cell wall damage conditions elicit rescue mechanisms to provide maintenance of cellular integrity and fungal survival. Through transcriptional reprogramming, yeast modulate the expression of genes important for cell wall biogenesis and remodeling, metabolism and energy generation, morphogenesis, signal transduction and stress. The yeast cell wall integrity (CWI) pathway, which is very well conserved in other fungi, is the key pathway for the regulation of this adaptive response. In this review, we summarize the current knowledge of the yeast transcriptional program elicited to counterbalance cell wall stress situations, the role of the CWI pathway in the regulation of this program and the importance of the transcriptional input received by other pathways. Modulation of this adaptive response through the CWI pathway by positive and negative transcriptional feedbacks is also discussed. Since all these regulatory mechanisms are well conserved in pathogenic fungi, improving our knowledge about them will have an impact in the developing of new antifungal therapies.Keywords: cell wall integrity; mitogen-activated protein kinase (MAPK); signal transduction; transcription; gene expression; antifungal Cell Wall Organization and Structure in S. cerevisiaeYeast cell integrity depends on the cell wall, an essential structure necessary not only for maintaining morphology but also for protecting cells against environmental stress conditions [1,2]. The cell wall is a macromolecular complex mainly composed of β-1,3-glucan, β-1,6-glucan, chitin and mannoproteins. Chitin, although a minor component, is essential for cell survival. β-1,3-glucan is the most abundant component of the yeast cell wall and serves as a backbone to which the other cell wall components are linked. The reducing ends of β-1,6-glucan and chitin are attached to the non-reducing end of β-1,3-glucan chains by an uncharacterized link and a β-1,4-linkage, respectively [3][4][5]. β-1,6-glucan is also attached to the chitin through β-1,3-linked oligogluco-residues that branch off the glucan. There is also a fraction of free chitin. Cell wall mannoproteins, including those involved in adhesion, cell wall remodeling, structural proteins and somatic antigens [6][7][8][9] can be linked directly to the β-1,3-glucan via alkali-labile bonds [9,10] and indirectly via β-1,6-glucan through a glycosylphosphatidylinositol (GPI) anchor [9,11]. The structure of the fungal cell wall is very well conserved among fungi despite several differences in cell wall composition exist [12]. Among fungal glucans, diversities in configuration, position of glyosidic bonds and branching have been reported [13]. β-1,3-glucan is the major cell wall component and is present in all the fungal species analyzed, whereas other polysaccharides including β-1,3/β-1,4-glucan, β-1,6-glucan or α-1,3-glucan a...

SummaryThe cross-linking of polysaccharides to assemble new cell wall in fungi requires transglycosylation mechanisms by which preexisting glycosidic linkages are broken and new linkages are created between the polysaccharides. The molecular mechanisms for these processes, which are essential for fungal cell biology, are only now beginning to be elucidated. Recent development of in vivo and in vitro biochemical approaches has allowed characterization of important aspects about the formation of chitin-glucan covalent cell wall cross-links by cell wall transglycosylases of the CRH family and their biological function. Covalent linkages between chitin and glucan mediated by Crh proteins control morphogenesis and also play important roles in the remodeling of the fungal cell wall as part of the compensatory responses necessary to counterbalance cell wall stress. These enzymes are encoded by multigene families of redundant proteins very well conserved in fungal genomes but absent in mammalian cells. Understanding the molecular basis of fungal adaptation to cell wall stress through these and other cell wall remodeling enzymatic activities offers an opportunity to explore novel antifungal treatments and to identify potential fungal virulence factors.

Crh proteins catalyze crosslinking of chitin and glucan polymers in fungal cell walls. Here, we show that the BcCrh1 protein from the phytopathogenic fungus Botrytis cinerea acts as a cytoplasmic effector and elicitor of plant defense. BcCrh1 is localized in vacuoles and the endoplasmic reticulum during saprophytic growth. However, upon plant infection, the protein accumulates in infection cushions; it is then secreted to the apoplast and translocated into plant cells, where it induces cell death and defense responses. Two regions of 53 and 35 amino acids are sufficient for protein uptake and cell death induction, respectively. BcCrh1 mutant variants that are unable to dimerize lack transglycosylation activity, but are still able to induce plant cell death. Furthermore, Arabidopsis lines expressing the bccrh1 gene exhibit reduced sensitivity to B. cinerea, suggesting a potential use of the BcCrh1 protein in plant immunization against this necrotrophic pathogen.

Summary O-mannosylation is a crucial protein modification in eukaryotes that is initiated by the essential family of protein O-mannosyltransferases (PMTs). Here we demonstrate that in the model yeast

The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway.

The Small World Initiative (SWI) and Tiny Earth are a consolidated and successful education programs rooted in the USA that tackle the antibiotic crisis by a crowdsourcing strategy. Based on active learning, it challenges young students to discover novel bioactive-producing microorganisms from environmental soil samples. Besides its pedagogical efficiency to impart microbiology content in academic curricula, SWI promotes vocations in research and development in Experimental Sciences and, at the same time, disseminates the antibiotic awareness guidelines of the World Health Organization. We have adapted the SWI program to the Spanish academic environment by a pioneering hierarchic strategy based on service-learning that involves two education levels (higher education and high school) with different degrees of responsibility. Throughout the academic year, 23 SWI teams, each consisting of 3-7 undergraduate students led by one faculty member, coordinated off-campus programs in 22 local high schools, involving 597 high school students as researchers. Post-survey-based evaluation of the program reveals a satisfactory achievement of goals: acquiring scientific abilities and general or personal competences by university students, as well as promoting academic decisions to inspire vocations for science- and technology-oriented degrees in younger students, and successfully communicating scientific culture in antimicrobial resistance to a young stratum of society.