Methods that shaped telomerase research.

Telomerase, the ribonucleoprotein (RNP) responsible for telomere maintenance, has a complex life. Complex in that it is made of multiple proteins and an RNA, and complex because it undergoes many changes, and passes through different cell compartments. As such, many methods have been developed to discover telomerase components, delve deep into understanding its structure and function and to figure out how telomerase biology ultimately relates to human health and disease. While some old gold-standard methods are still key for determining telomere length and measuring telomerase activity, new technologies are providing promising new ways to gain detailed information that we have never had access to before. Therefore, we thought it timely to briefly review the methods that have revealed information about the telomerase RNP and outline some of the remaining questions that could be answered using new methodology.

Maturation and shuttling of the yeast telomerase RNP: assembling something new using recycled parts.

As the limiting component of the budding yeast telomerase, the Tlc1 RNA must undergo multiple consecutive modifications and rigorous quality checks throughout its lifecycle. These steps will ensure that only correctly processed and matured molecules are assembled into telomerase complexes that subsequently act at telomeres. The complex pathway of Tlc1 RNA maturation, involving 5'- and 3'-end processing, stabilisation and assembly with the protein subunits, requires at least one nucleo-cytoplasmic passage. Furthermore, it appears that the pathway is tightly coordinated with the association of various and changing proteins, including the export factor Xpo1, the Mex67/Mtr2 complex, the Kap122 importin, the Sm7 ring and possibly the CBC and TREX-1 complexes. Although many of these maturation processes also affect other RNA species, the Tlc1 RNA exploits them in a new combination and, therefore, ultimately follows its own and unique pathway. In this review, we highlight recent new insights in maturation and subcellular shuttling of the budding yeast telomerase RNA and discuss how these events may be fine-tuned by the biochemical characteristics of the varying processing and transport factors as well as the final telomerase components. Finally, we indicate outstanding questions that we feel are important to be addressed for a complete understanding of the telomerase RNA lifecycle and that could have implications for the human telomerase as well.

Evaluating the Cytometric Detection and Enumeration of the Wine Bacterium, Oenococcus oeni.

Flow cytometry is a high-throughput tool for determining microbial abundance in a range of medical, environmental, and food-related samples. For wine, determining the abundance of Saccharomyces cerevisiae is well-defined and reliable. However, for the most common wine bacterium, Oenococcus oeni, using flow cytometry to determine cell concentration poses some challenges. O. oeni most often occurs in doublets or chains of varying lengths that can be greater than seven cells. This wine bacterium is also small, at 0.2-0.6 µm and may exhibit a range of morphologies including binary fission and aggregated complexes. This work demonstrates a straightforward approach to determining the suitability of flow cytometry for the chain-forming bacteria, O. oeni, and considerations when using flow cytometry for the enumeration of small microorganisms (<0.5 µm). © 2020 International Society for Advancement of Cytometry.

QTL mapping: an innovative method for investigating the genetic determinism of yeast-bacteria interactions in wine.

The two most commonly used wine microorganisms, Saccharomyces cerevisiae yeast and Oenococcus oeni bacteria, are responsible for completion of alcoholic and malolactic fermentation (MLF), respectively. For successful co-inoculation, S. cerevisiae and O. oeni must be able to complete fermentation; however, this relies on compatibility between yeast and bacterial strains. For the first time, quantitative trait loci (QTL) analysis was used to elucidate whether S. cerevisiae genetic makeup can play a role in the ability of O. oeni to complete MLF. Assessment of 67 progeny from a hybrid S. cerevisiae strain (SBxGN), co-inoculated with a single O. oeni strain, SB3, revealed a major QTL linked to MLF completion by O. oeni. This QTL encompassed a well-known translocation, XV-t-XVI, that results in increased SSU1 expression and is functionally linked with numerous phenotypes including lag phase duration and sulphite export and production. A reciprocal hemizygosity assay was performed to elucidate the effect of the gene SSU1 in the SBxGN background. Our results revealed a strong effect of SSU1 haploinsufficiency on O. oeni's ability to complete malolactic fermentation during co-inoculation and pave the way for the implementation of QTL mapping projects for deciphering the genetic bases of microbial interactions. KEY POINTS: • For the first time, QTL analysis has been used to study yeast-bacteria interactions. • A QTL encompassing a translocation, XV-t-XVI, was linked to MLF outcomes. • S. cerevisiae SSU1 haploinsufficiency positively impacted MLF by O. oeni.

The microbial challenge of winemaking: yeast-bacteria compatibility.

The diversity and complexity of wine environments present challenges for predicting success of fermentation. In particular, compatibility between yeast and lactic acid bacteria is affected by chemical and physical parameters that are strain and cultivar specific. This review focuses on the impact of compound production by microbes and physical interactions between microbes that ultimately influence how yeast and bacteria may work together during fermentation. This review also highlights the importance of understanding microbial interactions for yeast-bacteria compatibility in the wine context.

Measures to improve wine malolactic fermentation.

This review focuses on the considerable amount of research that has been directed towards the improvement of efficiency and reliability of malolactic fermentation (MLF), which is important in winemaking. From this large body of work, it is clear that reliable MLF is essential for process efficiency and prevention of spoilage in the final product. Impediments to successful MLF in wine, the impact of grape and wine ecology and how this may affect MLF outcome are discussed. Further focus is given to how MLF success may be enhanced, via alternative inoculation strategies, MLF progress sensing technologies and the use of different bacterial species. An update of how this information may be used to enhance and improve sensory outcomes through metabolite production during MLF and suggestions for future research priorities for the field are also provided.

Effects of ovarian disaggregation on adult murine follicle yield and viability.

Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 folliclesmg-1. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n=7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590UmL-1) or animal origin-free collagenase IV (AOF) at 590 or 1180UmL-1. Isolated follicles were stained with 4',6'-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17±10 folliclesmg-1 ovarian tissue). All disaggregation methods released more secondary follicles (86±20 per ovary; P<0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P<0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P<0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.

A mechanism for telomere-specific telomere length regulation.

Telomeric DNA, composed of short, direct repeats, is of crucial importance for chromosome stability. Due to intrinsic problems with replicating this DNA, the repeat tracts shorten at each cell division. Once repeat tracts become critically short, a telomeric stress signal induces cellular senescence and division arrest, which eventually may lead to devastating age-related degenerative diseases associated with dysfunctional telomers. Conversely, maintenance of telomere length by telomerase upregulation is a hallmark of cancer. Therefore, telomere length is a critical determinant of telomere function. How telomere length is established and molecular mechanisms for telomere-specific length regulation remained unknown. Here we show that subtelomeric chromatin is a determinant for how telomere equilibrium set-length is established in cis. The results demonstrate that telomerase recruitment mediated by the telomere-associated Sir4 protein is modulated on chromosome 3L in a telomere-specific way. Increased Sir4 abundance on subtelomeric heterochromatin of this specific telomere leads to telomere lengthening of only that telomere in cis, but not at other telomeres. Therefore, this work describes a mechanism for a how telomere-specific repeat tract length can be established. Further, our results will force the evaluation of telomere length away from a generalized view to a more telomere-specific consideration.

Ratcheted transport and sequential assembly of the yeast telomerase RNP.

The telomerase ribonucleoprotein particle (RNP) replenishes telomeric DNA and minimally requires an RNA component and a catalytic protein subunit. However, telomerase RNP maturation is an intricate process occurring in several subcellular compartments and is incompletely understood. Here, we report how the co-transcriptional association of key telomerase components and nuclear export factors leads to an export-competent, but inactive, RNP. Export is dependent on the 5' cap, the 3' extension of unprocessed telomerase RNA, and protein associations. When the RNP reaches the cytoplasm, an extensive protein swap occurs, the RNA is trimmed to its mature length, and the essential catalytic Est2 protein joins the RNP. This mature and active complex is then reimported into the nucleus as its final destination and last processing steps. The irreversible processing events on the RNA thus support a ratchet-type model of telomerase maturation, with only a single nucleo-cytoplasmic cycle that is essential for the assembly of mature telomerase.

Empirical parameterisation and dynamical analysis of the allometric Rosenzweig-MacArthur equations.

Allometric settings of population dynamics models are appealing due to their parsimonious nature and broad utility when studying system level effects. Here, we parameterise the size-scaled Rosenzweig-MacArthur differential equations to eliminate prey-mass dependency, facilitating an in depth analytic study of the equations which incorporates scaling parameters' contributions to coexistence. We define the functional response term to match empirical findings, and examine situations where metabolic theory derivations and observation diverge. The dynamical properties of the Rosenzweig-MacArthur system, encompassing the distribution of size-abundance equilibria, the scaling of period and amplitude of population cycling, and relationships between predator and prey abundances, are consistent with empirical observation. Our parameterisation is an accurate minimal model across 15+ orders of mass magnitude.