Antifungal activity of non-conventional yeasts against Botrytis cinerea and non-Botrytis grape bunch rot fungi.

Grapes harbour a plethora of non-conventional yeast species. Over the past two decades, several of the species have been extensively characterised and their contribution to wine quality is better understood. Beyond fermentation, some of the species have been investigated for their potential as alternative biological tools to reduce grape and wine spoilage. However, such studies remain limited to a few genera. This work aimed to evaluate the antagonistic activity of grape must-derived non-conventional yeasts against Botrytis cinerea and non-Botrytis bunch-rotting moulds and to further elucidate mechanisms conferring antifungal activity. A total of 31 yeast strains representing 21 species were screened on different agar media using a dual culture technique and liquid mixed cultures, respectively. Pichia kudriavzevii was the most potent with a minimum inhibitory concentration of 102 cells/mL against B. cinerea but it had a narrow activity spectrum. Twelve of the yeast strains displayed broad antagonistic activity, inhibiting three strains of B. cinerea (B05. 10, IWBT FF1 and IWBT FF2), a strain of Aspergillus niger and Alternaria alternata. Production of chitinases and glucanases in the presence of B. cinerea was a common feature in most of the antagonists. Volatile and non-volatile compounds produced by antagonistic yeast strains in the presence of B. cinerea were analysed and identified using gas and liquid chromatography mass spectrometry, respectively. The volatile compounds identified belonged mainly to higher alcohols, esters, organosulfur compounds and monoterpenes while the non-volatile compounds were cyclic peptides and diketopiperazine. To our knowledge, this is the first report to demonstrate inhibitory effect of the non-volatile compounds produced by various yeast species.

Distinct structural groups of histone H3 and H4 residues have divergent effects on chronological lifespan in Saccharomyces cerevisiae.

We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the transcriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism.

How Does Inflammation-Induced Hyperglycemia Cause Mitochondrial Dysfunction in Immune Cells?

Inflammatory mediators have an established role in inducing insulin resistance and promoting hyperglycemia. In turn, hyperglycemia has been argued to drive immune cell dysfunction as a result of mitochondrial dysfunction. Here, the authors review the evidence challenging this view. First, it is pointed out that inflammatory mediators are known to induce altered mitochondrial function. In this regard, critical care patients suffer both an elevated inflammatory tone as well as hyperglycemia, rendering it difficult to distinguish between the effects of inflammation and hyperglycemia. Second, emerging evidence indicates that a decrease in mitochondrial respiration and an increase in reactive oxygen species (ROS) production are not necessarily manifestations of pathology, but adaptations taking shape as the mitochondria is abdicating its adenosine triphosphate (ATP)-producing function (which is taken over by glycolysis) and instead becomes "retooled" for an immunological role. Collectively, these observations challenge the commonly held belief that acute hyperglycemia induces mitochondrial damage leading to immune cell dysfunction.

The human transketolase-like proteins TKTL1 and TKTL2 are bona fide transketolases.

BACKGROUND: Three transketolase genes have been identified in the human genome to date: transketolase (TKT), transketolase-like 1 (TKTL1) and transketolase-like 2 (TKTL2). Altered TKT functionality is strongly implicated in the development of diabetes and various cancers, thus offering possible therapeutic utility. It will be of great value to know whether TKTL1 and TKTL2 are, similarly, potential therapeutic targets. However, it remains unclear whether TKTL1 and TKTL2 are functional transketolases. RESULTS: Homology modelling of TKTL1 and TKTL2 using TKT as template, revealed that both TKTL1 and TKTL2 could assume a folded structure like TKT. TKTL1/2 presented a cleft of suitable dimensions between the homodimer surfaces that could accommodate the co-factor-substrate. An appropriate cavity and a hydrophobic nodule were also present in TKTL1/2, into which the diphosphate group fitted, and that was implicated in aminopyrimidine and thiazole ring binding in TKT, respectively. The presence of several identical residues at structurally equivalent positions in TKTL1/2 and TKT identified a network of interactions between the protein and co-factor-substrate, suggesting the functional fidelity of TKTL1/2 as transketolases. CONCLUSIONS: Our data support the hypothesis that TKTL1 and TKTL2 are functional transketolases and represent novel therapeutic targets for diabetes and cancer.

Well-positioned nucleosomes punctuate polycistronic pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei.

BACKGROUND: The compaction of DNA in chromatin in eukaryotes allowed the expansion of genome size and coincided with significant evolutionary diversification. However, chromatin generally represses DNA function, and mechanisms coevolved to regulate chromatin structure and its impact on DNA. This included the selection of specific nucleosome positions to modulate accessibility to the DNA molecule. Trypanosoma brucei, a member of the Excavates supergroup, falls in an ancient evolutionary branch of eukaryotes and provides valuable insight into the organization of chromatin in early genomes. RESULTS: We have mapped nucleosome positions in T. brucei and identified important differences compared to other eukaryotes: The RNA polymerase II initiation regions in T. brucei do not exhibit pronounced nucleosome depletion, and show little evidence for defined -1 and +1 nucleosomes. In contrast, a well-positioned nucleosome is present directly on the splice acceptor sites within the polycistronic transcription units. The RNA polyadenylation sites were depleted of nucleosomes, with a single well-positioned nucleosome present immediately downstream of the predicted sites. The regions flanking the silent variant surface glycoprotein (VSG) gene cassettes showed extensive arrays of well-positioned nucleosomes, which may repress cryptic transcription initiation. The silent VSG genes themselves exhibited a less regular nucleosomal pattern in both bloodstream and procyclic form trypanosomes. The DNA replication origins, when present within silent VSG gene cassettes, displayed a defined nucleosomal organization compared with replication origins in other chromosomal core regions. CONCLUSIONS: Our results indicate that some organizational features of chromatin are evolutionarily ancient, and may already have been present in the last eukaryotic common ancestor.

Bioinformatics tools for the structural elucidation of multi-subunit protein complexes by mass spectrometric analysis of protein-protein cross-links.

Multi-subunit protein complexes are involved in many essential biochemical processes including signal transduction, protein synthesis, RNA synthesis, DNA replication and protein degradation. An accurate description of the relative structural arrangement of the constituent subunits in such complexes is crucial for an understanding of the molecular mechanism of the complex as a whole. Many complexes, however, lie in the mega-Dalton range, and are not amenable to X-ray crystallographic or nuclear magnetic resonance analysis. Techniques that are suited to structural studies of such large complexes, such as cryo-electron microscopy, do not provide the resolution required for a mechanistic insight. Mass spectrometry (MS) has increasingly been applied to identify the residues that are involved in chemical cross-links in compound protein assemblies, and have provided valuable insight into the molecular arrangement, orientation and contact surfaces of subunits within such large complexes. This approach is known as MS3D, and involves the MS analysis of cross-linked di-peptides following the enzymatic cleavage of a chemically cross-linked complex. A major challenge of this approach is the identification of the cross-linked di-peptides in a composite mixture of peptides, as well as the identification of the residues involved in the cross-link. These analyses require bioinformatics tools with capabilities beyond that of general, MS-based proteomic analysis software. Many MS3D software tools have appeared, often designed for very specific experimental methods. Here, we provide a review of all major MS3D bioinformatics programmes, reviewing their applicability to different workflows, specific experimental requirements and the computational approach taken by each.