Thermosonication as an alternative method for processing, extending the shelf life, and conserving the quality of pulque: A non-dairy Mexican fermented beverage.

The aim of this study was to evaluate thermosonication as an alternative method for the pasteurization of pulque in order to improve its shelf life and retain its quality parameters. Thermosonication was carried out at 50 °C using amplitudes of 75% (for 6 and for 9 min), 85% (for 4 and for 6 min), and 95% (for 3 and for 5 min). These were the optimal conditions found for processing pulque by thermosonication. Physicochemical (acidity, color, alcohol content, and sensory analysis) and microbiological (lactic acid bacteria and yeasts) parameters were determined during 30 days for storage at 4 ± 1 °C. Conventional pasteurization (63 °C, 30 min) and raw pulque were used as controls. According to the results, the shelf life of pulque was extended up to 24 days storage at 4 °C. After this time, the quality of beverage decreased, due that the microbial load increases. Thermosonication treatments at 75% and 85% showed a higher content of LAB (6.58-6.77 log CFU/mL) and yeasts (7.08-7.27 log CFU/mL) than conventional pasteurization (3.64 log CFU/mL of LAB and 3.97 log CFU/mL of yeasts) at 24 days of storage. Raw pulque demonstrated up to 7.77 log CFU/mL of yeasts and 7.51 log CFU/mL of LAB. Pulque processed by thermosonication exhibited greater lightness, sensory acceptance, a maximal acidity of 0.83 g/lactic acid, and an alcohol content of 4.48-4.95% v/v. The thermosonication process preserves sensory and physicochemical properties better than conventional pasteurization. Lactic acid bacteria such as Lactobacillus kefiri, Lactobacillus acidophilus, and Lactobacillus hilgardii and yeasts such as Saccharomyces cereviasiae were identified in thermosonicated pulque.

An appreciation of the prescience of Don Gilbert (1930-2011): master of the theory and experimental unravelling of biochemical and cellular oscillatory dynamics.

We review Don Gilbert's pioneering seminal contributions that both detailed the mathematical principles and the experimental demonstration of several of the key dynamic characteristics of life. Long before it became evident to the wider biochemical community, Gilbert proposed that cellular growth and replication necessitate autodynamic occurrence of cycles of oscillations that initiate, coordinate and terminate the processes of growth, during which all components are duplicated and become spatially re-organised in the progeny. Initiation and suppression of replication exhibit switch-like characteristics, that is, bifurcations in the values of parameters that separate static and autodynamic behaviour. His limit cycle solutions present models developed in a series of papers reported between 1974 and 1984, and these showed that most or even all of the major facets of the cell division cycle could be accommodated. That the cell division cycle may be timed by a multiple of shorter period (ultradian) rhythms, gave further credence to the central importance of oscillatory phenomena and homeodynamics as evident on multiple time scales (seconds to hours). Further application of the concepts inherent in limit cycle operation as hypothesised by Gilbert more than 50 years ago are now validated as being applicable to oscillatory transcript, metabolite and enzyme levels, cellular differentiation, senescence, cancerous states and cell death. Now, we reiterate especially for students and young colleagues, that these early achievements were even more exceptional, as his own lifetime's work on modelling was continued with experimental work in parallel with his predictions of the major current enterprises of biological research.

Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B.

Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 µg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

The oesophageal diverticulum of Dirioxa pornia studied through micro-CT scan, dissection and SEM studies.

BACKGROUND: Dirioxa pornia (Diptera, Tephritidae) (Island fly) is an Australian native species related to a number of pestiferous fruit flies but, unlike many of the pest species, has not been studied extensively due to its non-pest status. However, due to D. pornia's apparent reliance on the bacteria for survival it is an ideal species to undertake studies into interaction between Tephritid species and bacteria associated with the intestinal tract. The oesophageal diverticulum, which is a blind-ended protrusion of the oesophagus, has been studied, described and characterised in many other Tephritid species. Unlike many other species where the oesophageal diverticulum has been observed the organ was only observed in male D. pornia. It is speculated that this sexual dimorphism the organ may be the primary location to host beneficial bacteria in the involved in the production of the nuptial gift and the mating success of this Tephritid species. In case of D. pornia, however, no study on any area of the digestive system has been conducted. This study was conducted to locate and characterize the oesophageal diverticulum in D. pornia. A virtual dissection of the alimentary tract was made through micro-computer tomography studies. These studies were followed by dissection and scanning microscopy studies to elucidate the presence of bacteria. RESULTS: The oesophageal diverticulum of D. pornia is part of the foregut and distends from the oesophagus within the head of the fly. The shape of the oesophageal diverticulum corresponds with the Ceratitis type. Scanning microscopy studies of the oesophageal diverticulum show rod-shaped bacterial cells residing along with yeast cells in the lumen. The organ was only observed in male specimens. CONCLUSIONS: This study classifies the oesophageal diverticulum of D. pornia under the "Ceratitis type" of oesophageal diverticula in Tephritid species. The study also proves that micro-CT scanning is possible to locate soft tissues in Tephritid species and the Avizo® Fire software can be successfully used to visualize 3 dimensional (3D) images from x-rays. The methods used in this experiment can be used in future studies for visualising soft tissues of adult Tephritid species through micro tomography. There is sexual dimorphism with the organ only found in males. Finally this study shows that bacteria are present in the oesophageal diverticulum of D. pornia.

INDIANA: An in-cell diffusion method to characterize the size, abundance and permeability of cells.

NMR and MRI diffusion experiments contain information describing the shape, size, abundance, and membrane permeability of cells although extracting this information can be challenging. Here we present the INDIANA (IN-cell DIffusion ANAlysis) method to simultaneously and non-invasively measure cell abundance, effective radius, permeability and intrinsic relaxation rates and diffusion coefficients within the inter- and intra-cellular populations. The method couples an experimental dataset comprising stimulated-echo diffusion measurements, varying both the gradient strength and the diffusion delay, together with software to fit a model based on the Kärger equations to robustly extract the relevant parameters. A detailed error analysis is presented by comparing the results from fitting simulated data from Monte Carlo simulations, establishing its effectiveness. We note that for parameters typical of mammalian cells the approach is particularly effective, and the shape of the underlying cells does not unduly affect the results. Finally, we demonstrate the performance of the experiment on systems of suspended yeast and mammalian cells. The extracted parameters describing cell abundance, size, permeability and relaxation are independently validated.

Yeast peroxisomes: How are they formed and how do they grow?

Peroxisomes are single membrane enclosed cell organelles, which are present in almost all eukaryotic cells. In addition to the common peroxisomal pathways such as ß-oxidation of fatty acids and decomposition of H2O2, these organelles fulfil a range of metabolic and non-metabolic functions. Peroxisomes are very important since various human disorders exist that are caused by a defect in peroxisome function. Here we describe our current knowledge on the molecular mechanisms of peroxisome biogenesis in yeast, including peroxisomal protein sorting, organelle dynamics and peroxisomal membrane contact sites.

Onychomycosis due to dermatophytes species in Iran: Prevalence rates, causative agents, predisposing factors and diagnosis based on microscopic morphometric findings.

OBJECTIVE: Onychomycosis (OM) or fungal nail infection is one of the most common fungal infections, which is increasingly prevalent. OM is caused by dermatophytes spp, yeasts and non-dermatophyte moulds (NDMs). The purpose of this study was to identify and determine the prevalence rates, predisposing factors and causative agents of OM using clinical symptoms and microscopic morphometric findings. MATERIALS AND METHODS: In the present study, 180 patients suspected of OM were evaluated by direct microscopy using KOH 20%, culturing in Mycosel and Sabouraud dextrose agar media and Olysia software for identifying the causative fungi of OM. RESULTS: From 180 referred patients, 118 (65.56%) had OM, of whom 79 (66.94%) were positive for infection with dermatophytes spp. Of the 79 cases, the commonest age group was 61-70 years (21%) with males being 46 (58.23%) and females being 33 (41.77%). Both the fingernail and toenail infections were most prevalent in male patients. Sex, diabetes and age above 60 years were significant predisposing factors for OM development. DLSO was observed as the only clinical pattern of OM and T. rubrum was the commonest dermatophyte isolate (49.34%). CONCLUSION: This study showed that T. rubrum was the most common dermatophyte agent of OM in Iran.

High-resolution structures of mitochondrial ribosomes and their functional implications.

Mitochondrial ribosomes (mitoribosomes) almost exclusively synthesize essential components of the oxidative phosphorylation machinery. Dysfunction of mitochondrial protein biosynthesis leads to human diseases and plays an important role in the altered metabolism of cancer cells. Recent developments in cryo-electron microscopy enabled the structural characterization of complete yeast and mammalian mitoribosomes at near-atomic resolution. Despite originating from ancestral bacterial ribosomes, mitoribosomes have diverged in their composition and architecture. Mitoribosomal proteins are larger and more numerous, forming an extended network around the ribosomal RNA, which is expanded in yeast and highly reduced in mammals. Novel protein elements at the entrance or exit of the mRNA channel imply a different mechanism of mRNA recruitment. The polypeptide tunnel is optimized for the synthesis of hydrophobic proteins and their co-translational membrane insertion.

Yeast-like microorganisms in the scale insect Kermes quercus (Insecta, Hemiptera, Coccomorpha: Kermesidae). Newly acquired symbionts?

Scale insects, like other plant sap-consumers, are host to symbiotic microorganisms which provide them with the substances missing from their diet. In contrast to most scale insects, Kermes quercus (Linnaeus) was regarded as asymbiotic. Our histological and ultrastructural observations show that in the body of the feeding stages of K. quercus collected in two locations (Warsaw and Cracow), numerous yeast-like microorganisms occur. These microorganisms were localized in the cytoplasm of fat body cells. The yeast-like microorganisms were observed neither in other organs of the host insect nor in the eggs. These microorganisms did not cause any damage to the structure of the ovaries and the course of oogenesis of the host insect. The females infected by them produced about 1300 larvae. The lack of these microorganisms in the cytoplasm of eggs indicates that they are not transmitted transovarially from mother to offspring. Molecular analyses indicated that the microorganisms which reside in the body of K. quercus are closely related to the entomopathogenic fungi Cordyceps and Ophiocordyceps, which belong to the Sordariomycetes class within the Ascomycota. The role of yeast-like microorganisms to their host insects remains unknown; however, it has been suggested that they may represent newly acquired symbionts.

Common lines modeling for reference free Ab-initio reconstruction in cryo-EM.

We consider the problem of estimating an unbiased and reference-free ab initio model for non-symmetric molecules from images generated by single-particle cryo-electron microscopy. The proposed algorithm finds the globally optimal assignment of orientations that simultaneously respects all common lines between all images. The contribution of each common line to the estimated orientations is weighted according to a statistical model for common lines' detection errors. The key property of the proposed algorithm is that it finds the global optimum for the orientations given the common lines. In particular, any local optima in the common lines energy landscape do not affect the proposed algorithm. As a result, it is applicable to thousands of images at once, very robust to noise, completely reference free, and not biased towards any initial model. A byproduct of the algorithm is a set of measures that allow to asses the reliability of the obtained ab initio model. We demonstrate the algorithm using class averages from two experimental data sets, resulting in ab initio models with resolutions of 20Å or better, even from class averages consisting of as few as three raw images per class.

Role of Intra- and Inter-mitochondrial Membrane Contact Sites in Yeast Phospholipid Biogenesis.

Eukaryotic cells exhibit intracellular compartments called organelles wherein various specialized enzymatic reactions occur. Despite the specificity of the characteristic functions of organelles, recent studies have shown that distinct organelles physically connect and communicate with each other to maintain the integrity of their functions. In yeast, multiple inter- and intramitochondrial membrane contact sites (MCSs) were identified to date and were proposed to be involved in phospholipid biogenesis. In the present article, we focus on inter- and intra-organellar MCSs involving mitochondria and their tethering factors, such as the ERMES (endoplasmic reticulum (ER)-mitochondria encounter structure) complex and EMC (conserved ER membrane protein complex) between mitochondria and the ER, vCLAMP (vacuole and mitochondria patch) between mitochondria and vacuoles, and the MICOS (mitochondrial contact site) complex between the mitochondrial outer and inner membranes (MOM and MIM). All of these membrane-tethering factors were proposed to be involved in phospholipid biogenesis. Furthermore, the existence of functional interconnections among multiple organelle contact sites is suggested. In the present article, we summarize the latest discoveries in regard to MCSs and MCS-forming factors involving mitochondria and discuss their molecular functions, with particular focus on phospholipid metabolism in yeast.

Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole.

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole.

Mussel-Inspired Surface-Imprinted Sensors for Potentiometric Label-Free Detection of Biological Species.

Using sensors to quantify clinically relevant biological species has emerged as a fascinating research field due to their potential to revolutionize clinical diagnosis and therapeutic monitoring. Taking advantage of the wide utility in clinical analysis and low cost of potentiometric ion sensors, we demonstrate a method to use such ion sensors to quantify bioanalytes without chemical labels. This is achieved by combination of chronopotentiometry with a mussel-inspired surface imprinting technique. The biomimetic sensing method is based on a blocking mechanism by which the recognition reaction between the surface imprinted polymer and a bioanalyte can block the current-induced ion transfer of an indicator ion, thus causing a potential change. The present method offers high sensitivity and excellent selectivity for detection of biological analytes. As models, trypsin and yeast cells can be measured at levels down to 0.03 U mL-1 and 50 CFU mL-1 , respectively.

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy.

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning.

High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

Analysis of Yeast Mitochondria by Electron Microscopy.

Budding yeast Saccharomyces cerevisiae represents a widely used model organism for the study of mitochondrial biogenesis and architecture. Electron microscopy is an essential tool in the analysis of cellular ultrastructure and the precise localization of proteins to organellar subcompartments. We provide here detailed protocols for the analysis of yeast mitochondria by transmission electron microscopy: (1) chemical fixation and Epon embedding of yeast cells and isolated mitochondria, and (2) cryosectioning and immunolabeling of yeast cells and isolated mitochondria according to the Tokuyasu method.

The antileishmanial activity of xanthohumol is mediated by mitochondrial inhibition.

Xanthohumol (Xan) is a natural constituent of human nutrition. Little is known about its actions on leishmanial parasites and their mitochondria as putative target. Therefore, we determined the antileishmanial activity of Xan and resveratrol (Res, as alternative compound with antileishmanial activity) with respect to mitochondria in Leishmania amazonensis promastigotes/amastigotes (LaP/LaA) in comparison with their activity in peritoneal macrophages from mouse (PMM) and macrophage cell line J774A.1 (J774). Mechanistic studies were conducted in Leishmania tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Xan and Res demonstrated antileishmanial activity in LaA [half inhibitory concentration (IC50): Xan 7 µ m, Res 14 µ m]; while they had less influence on the viability of PMM (IC50: Xan 70 µ m, Res >438 µ m). In contrast to Res, Xan strongly inhibited oxygen consumption in Leishmania (LtP) but not in J774 cells. This was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan, which was less pronounced with Res. Neither Xan nor Res increased mitochondrial superoxide release in LtP, while both decreased the mitochondrial membrane potential in LtP. Bioenergetic studies showed that LtP mitochondria have no spare respiratory capacity in contrast to mitochondria in J774 cells and can therefore much less adapt to stress by mitochondrial inhibitors, such as Xan. These data show that Xan may have antileishmanial activity, which is mediated by mitochondrial inhibition.

Asymmetric Localization of Components and Regulators of the Mitotic Exit Network at Spindle Pole Bodies.

Most proteins of the Mitotic Exit Network (MEN) and their upstream regulators localize at spindle pole bodies (SPBs) at least in some stages of the cell cycle. Studying the SPB localization of MEN factors has been extremely useful to elucidate their biological roles, organize them in a hierarchical pathway, and define their dynamics under different conditions.Recruitment to SPBs of the small GTPase Tem1 and the downstream kinases Cdc15 and Mob1/Dbf2 is thought to be essential for Cdc14 activation and mitotic exit, while that of the upstream Tem1 regulators (the Kin4 kinase and the GTPase activating protein Bub2-Bfa1) is important for MEN inhibition upon spindle mispositioning. Here, we describe the detailed fluorescence microscopy procedures that we use in our lab to analyze the localization at SPBs of Mitotic Exit Network (MEN) components tagged with GFP or HA epitopes.