Viable Yeast Cells Research Articles

Analysis of Frozen Bakery Items Containing Trehalose and its Impact on Quantitative Quality Measures

The frozen dough market has been increasing annually since it reduces cost while ensuring the availability of fresh products. Ice crystal formation inside the dough decreases yeast functionality while thawing releases fluid inside the dough, both leading to an unacceptable product. Trehalose can enhance the viability of yeast cells and reduce ice crystals. Four concentrations of trehalose (0, 10, 20, and 30%) were investigated on the freeze-thaw quality of frozen dough. Samples were prepared, frozen, and thawed before testing for color, moisture, water activity, and texture. Trehalose had no significant impact on color, moisture, or water activity of any sample despite trends in moisture and water activity being present. However, increased trehalose showed decrease in peak load in biscuits and pizza crusts. Therefore, more research is needed but data shows that trehalose could act as an anti-staling agent by reducing crust hardness in frozen biscuits and pizza crusts.

Assessing the physiological properties of baker's yeast based on single-cell Raman spectrum technology

With rapid progress in the yeast fermentation industry, a comprehensive commercial yeast quality assessment approach integrating efficiency, accuracy, sensitivity, and cost-effectiveness is required. In this study, a new yeast quality assessment method based on single-cell Raman technology was developed and contrasted with traditional methods. The findings demonstrated significant associations (Pearson correlation coefficient of 0.933 on average) between the two methods in measuring physiological indicators, including cell viability and intracellular trehalose content, demonstrating the credibility of the Raman method compared to the traditional method. Furthermore, the sensitivity of the Raman method in viable but non-culturable cells was higher in measuring yeast cell viability (17.9 % more sensitive). According to the accurate quantitative analysis of metabolic activity level (MAL) of yeast cells, the cell vitality was accurately quantified at population and single-cell levels, offering a more comprehensive assessment of yeast fermentation performance. Overall, the single-cell Raman method integrates credibility, feasibility, accuracy, and sensitivity in yeast quality assessment, offering a new technological framework for quality assessments of live-cell yeast products.

Evaluation of different hydration techniques on the viability of commercial dry brewing yeast

The utilization of dry yeast is increasingly becoming established in the brewing context, owing to the ease of storage and handling, the long shelf life and the convenience of dispensing a delicate and costly propagation process for fresh liquid cultures, which would need specialized labor and equipment. Dried yeasts undergo a procedure that removes nearly all liquid contents from the cell, and rehydration is then recommended prior to inoculation into the wort. Hydration is a nuanced procedure that allows yeasts to regain their vitality and viability, however, if carried out improperly, it can adversely affect fermentation. This study aimed to evaluate the impact of four hydration techniques on the viability and vitality of dry commercial yeast cells, preliminarily evaluating the influence of hydration using distilled and filtered water. Two different methodologies were employed, including the assessment of viability, by counting live and dead cells in the Neubauer chamber and the evaluation of vitality, by measuring the CO₂ released after inoculation. The hydration techniques tested included hydration in water without stirring, hydration in water with stirring, and these two techniques replicated in dry malt extract (DME) wort with Specific Gravity (SG) 1040. Firstly, it was found that rehydrating the yeast in distilled water has a negative impact on cell viability. Also, the results indicated that initial agitation has a negative effect on yeast viability and vitality, while hydration in water without agitation produced the best results. Correct hydration of dry yeast is crucial for avoiding off-flavors due to accelerated or delayed fermentation.

Craft Beer Produced by Immobilized Yeast Cells with the Addition of Grape Pomace Seed Powder: Physico-Chemical Characterization and Antioxidant Properties.

The aim of this study was to produce and to characterize craft beer fermented by immobilized yeast cells with the addition of Prokupac grape pomace seed powder (2.5% and 5%), to obtain a beer enriched with phenolic compounds and improved sensory characteristics. The immobilization of the yeast cells was performed by electrostatic extrusion, while the obtained calcium alginate beads were characterized by light and scanning electron microscopy. Phenolic and hop-derived bitter compounds in beer with or without grape pomace seed powder (GS) phenolics were identified using UHPLC Q-ToF MS. The results indicated that GS adjunct significantly shortened the fermentation process of wort and increased the content of phenolic compounds, especially ellagic acid, flavan-3-ols and pro(antho)cyanidins in the final products compared to the control beer. A total of twenty (iso)-α-acids and one prenylflavonoid were identified, although their levels were significantly lower in beers with GS phenolics compared to the control beer. Beers with GS phenolics showed good antioxidant properties as measured by the reduction of ferric ions (FRP) and the scavenging of ABTS•+ and DPPH• radicals. The concentration of immobilized viable yeast cells was higher than 1 × 108 CFU/g wet mass after each fermentation without destroying the beads, indicating that they can be reused for the repeated fermentation of wort. Beers produced with 5% GS added to the wort exhibited the best sensory properties (acidity, astringency, bitterness intensity, mouthfeel, aftertaste and taste), and highest overall acceptability by the panelists. The results showed that grape pomace seed powder present a promising adjunct for the production of innovative craft beer with good sensory properties and improved functionality.

Fdo1, Fkh1, Fkh2, and the Swi6-Mbp1 MBF complex regulate Mcd1 levels to impact eco1 rad61 cell growth in Saccharomyces cerevisiae.

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature-sensitive (ts) growth defects exhibited by eco1rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.

Sources of Variability in Determinations of Brewing Yeast Total Cell Counts and Viability Obtained by Microscopy

Brewers utilizing yeast cell counts and determinations of yeast viability obtained by microscopy require precise results for calculating the yeast pitch to produce fermentations with the highest quality and consistency. The steps in obtaining results with the desired precision include identifying, quantifying and controlling the sources of variation. In this study, we used simulated cell counting, manual cell counting, and semi-automated cell counting to explore sources of variation contributing to the variability of yeast cell counts and yeast viability determinations. The results showed that the variability associated with counting different fields of view contributed most to the variability in yeast cell counts and determinations of yeast viability. Understanding the sources and amounts of variation in cell counts will help brewers direct their efforts toward obtaining the highest quality determinations of yeast cell counts and viability.

Pulsed light treatment helps reduce sulfur dioxide required to preserve Malbec wines

Sulfur dioxide (SO2) has a long history of use as a preservative in wine production to prevent oxidation and microbial spoilage. However, sulfite sensitivities exist in some consumers that range from mild breathing irregularities to more severe allergic symptoms that can include anaphylactic reactions. These adverse effects have inspired the wine industry to develop alternative approaches to either lower or replace the use of sulfites. This study focused on assessing the efficacy of employing continuous Pulsed Light (PL) in conjunction with reducing SO2 applications to inactivate spoilage microorganisms and ensure optimal red wine preservation. Applying PL at an intensity of 4 J/cm2 to wine in combination with a 25 mg/L SO2 treatment generated a 12% and 22% reduction, respectively, in both the number of viable yeast and LAB cells, respectively, while having no substantial effects on color, changes in acidity, total phenolic, and anthocyanin contents after four months of aging. We conclude that PL processing of wine can help reduce the amount of SO2 needed to inactivate wine spoilage microorganisms and prevent loss of wine quality due to chemical oxidation. This combination of markedly reducing SO2 concentration use along with introducing non-thermal PL processing can be done effectively and simply to ensure a safer and stable final wine product for sulfite-sensitive consumers.

Design and optimization of microchannel for enhancement of the intensity of induced signal in particle/cell impedance measurement.

The measured impedance signal of a single particle or cell in a microchannel is of the μA level, which is a challenge for measuring such weak signals. Therefore, it is necessary to improve the intensity for expanding the applications of impedance measurement. In this paper, we analyzed the impact of geometric parameters of microchannel on output signal intensity by using the three-dimensional finite element method. In comparison to conventional microchannels, which are distributed at a uniform height, the microchannels in this design use the height difference to enhance the signal intensity. By analyzing the effects of the geometric dimensions of the constriction channel, main channel height, radius of particles, types of cells, shapes of particles with different ellipticities, and particles spacing on the current signal, we concluded the optimal dimensions of these parameters to improve the intensity of the induced current signal. Through the fabrication of the optimized size of device and experimental demonstration, it is verified that the current signal intensity caused by the particle with a diameter of 10 µm is nearly twice that of the conventional structure with a height of 20 µm, which proves the correctness of the optimization results and the feasibility of this work. In addition, the performance of the device was verified by measuring the mixtures of different size particles as well as non-viable and viable yeast cells.

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

Simulated aging of draught beer line tubing increases biofilm contamination

Craft brewing is continually gaining popularity in the United States. Craft brewers are committed to producing a wide variety of products and have a vested interest in product quality. Therefore, these brewers have the expectation that the beer poured at the tap will match the quality product that left the brewery. The presence of biofilm in draught lines is hypothesized as a contributing factor when this expectation is not achieved. Clean in place strategies based on the Sinner's Circle of Cleaning are used to remediate organic and inorganic accumulation in beer draught lines, including controlling biofilm accumulation. A study was conducted to determine if repeated exposure to chemical cleaning of vinyl beer tubing impacted biofilm growth, kill/removal, and subsequent regrowth of a mixed species biofilm. The tubing was conditioned to simulate one, two, and five years of use. The data collected demonstrates a clear trend between simulated age of the tubing and biofilm accumulation on the surface. Bacterial log densities ranged from 5.6 Log10(CFU/cm2) for the new tubing to 6.6 Log10(CFU/cm2) for tubing aged to simulate five years of use. The counts for the yeast were similar. Caustic cleaning of the tubing, regardless of starting biofilm coverage, left less than 2.75 Log10(CFU/cm2) viable bacteria and yeast cells remaining on the tubing surface. This demonstrated the effectiveness of the caustic at controlling biofilm accumulation in the simulated beer draught line. The biofilm that accumulated in the five-year aged tubing was able to recover more quickly, reaching 3.6 Log10(CFU/cm2) within 24 h indicating the treatment did not fully eradicate the biofilm, suggesting that the strong chemistry used in this study would cease to be as effective over time.

Viability test of yeast encapsulation (Candida tropicalis) using sodium alginate polymer in bread production

This study explores sodium alginate encapsulation’s impact on Candida tropicalis yeast cell viability and its influence on bread making. Encapsulation protects yeast cells from damage during drying and storage. The research used 10% and 15% sodium alginate for two isolates and one control. Statistical analysis, including Kruskal Wallis and Mann-Whitney tests in Microsoft Excel SPSS, showed significant effects. Sodium alginate encapsulation notably improved Candida tropicalis yeast cell viability and final bread swelling, especially at 15% concentration. Mann-Whitney tests on organoleptic parameters revealed panelist preferences: Candida tropicalis 2 with 15% sodium alginate for color, taste, and texture, while Candida tropicalis 1 with 15% sodium alginate for aroma. This study suggests sodium alginate’s potential to enhance yeast viability and improve bread quality, offering insights for food processing and preservation applications. The research findings may contribute to developing effective encapsulation techniques for yeast-based products.

Separating live and dead yeast cells in a serpentine microchannel using Dielectrophoresis

Separation techniques and particle manipulation has always been a matter of curiosity to research groups worldwide for numerous biomedical applications like cell concentration, separation, patterning, and trapping. The present work describes the fabrication and testing for constant cell separation using Dielectrophoresis phenomenon. The cells with different electric properties separate continuously at different location as they flow across the channel. Known mixture of non viable and viable yeast cells is selectively trapped using negative Dielectrophoresis. We achieved the cell trapping efficiency of 98%. A serpentine structure in the cell separation device appears to be a design choice that addresses the need for continuous separation and improved efficiency by minimizing dead volume.

Shaping the chromatic characteristics of red wines by using biofilm-detached cells of Starmerella bacillaris strains

The aim of this study was to evaluate the effects of 10 Starmerella bacillaris strains inoculated as planktonic or biofilm-detached cells on the chromatic characteristics of Montepulciano d’Abruzzo wine. Wines inoculated with biofilm-detached cells of St. bacillaris were characterized by a higher content of glycerol and viable yeast cells and a lower content of ethanol than those obtained with planktonic cells. Pyruvic acid content ranged from 45.99 mg/L to 48.19 mg/L and from 41.13 mg/L to 45.9 mg/L in wines fermented with biofilm-detached and planktonic cells, respectively. Wines obtained with biofilm-detached cells showed levels of anthocyanins ranging from 506.8 mg/L to 659.9 mg/L, while those fermented with free cells of St. bacillaris ranged from 518 mg/L to 612.6 mg/L. Similarly, the content of polyphenols was higher in wines inoculated with biofilm-detached cells. The different amounts of these compounds resulted in differences in the wine's color. Wines obtained with biofilm-detached cells of St. bacillaris had lower b* and h* values than those obtained with planktonic cells. These wines also showed higher a* values, indicating the presence of a stronger red color than the others, and lower clarity (L*). Moreover, the data obtained highlighted that it is possible to predict the color of young wines from must measurements. Further studies will be done to evaluate the role of other non-Saccharomyces yeasts, grown under different aggregation states, in the definition of wine color.

Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis.

Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis.

Yolk-Shell Encapsulation of Cells by Biomimetic Mineralization and Visible Light-Induced Surface Graft Polymerization.

The pursuit of low-cytotoxicity modification strategies represents a prominent avenue in cell coating research, holding immense significance for the advancement of practical living cell-related technologies. Here, we presented a novel method to fabricate encapsulated yeast cells with a yolk-shell structure by biomimetic mineralization and visible-light-induced surface graft polymerization. In this approach, an amorphous calcium carbonate (ACC) shell was first deposited on the surface of a yeast cell (cell@ACC) modified with 4 layers of self-assembled poly(diallyl dimethylammonium chloride) (PDADMAC)/poly(acrylic acid) (PAA) film using a biomimetic mineralization technique. Subsequently, polyethylenimine (PEI) was absorbed on the surface of cell@ACC by electrostatic interaction. Then, a cross-linked shell was introduced by surface-initiated graft polymerization of poly(ethylene glycol) diacrylate (PEGDA) on cell@ACC under irradiation of visible light using thioxanthone catechol-O,O'-diacetic acid as the photosensitizer. After the removal of the inner ACC shell, the yolk-shell-structured yeast cells (cell@PHS) were obtained. Due to the mild conditions of the strategy, the cell@PHS could retain 98.81% of its original viability. The introduction of the shell layer significantly prolonged the lag phase of yeast cells, which could be tuned between 5 and 25 h by regulating the thickness of the shell. Moreover, the cell@PHS showed improved resistance against lyticase due to the presence of a protective shell. After 30 days of storage, the viability of cell@PHS was 81.09%, which is significantly higher than the 19.89% viability of native yeast cells.

Using dielectrophoretic spectra to identify and separate viable yeast cells.

Immotile yeast cells were transiently moved in nonuniform sinusoidal electric fields using multiple pairs of micro-parallel cylindrical electrodes equipped with a sequential signal generator (SSG) to analyze cell viability at a clinical scale for the brewery/fermentation industry. Living yeast cells of Saccharomyces cerevisiae during the exponential-stationary phase, with a cell density of 1.15 × 105 cells mL-1 were suspended in sucrose medium. The conductivity (σs) was adjusted to 0.01 S m-1 with added KCl. Cells exposed in electric field strengths ranging from 32.89 to 40.98 kV m-1, exhibited positive dielectrophoresis (pDEP) with the lower critical frequencies (LCF) at 85.72 ± 3.59 kHz. The optimized value of LCF was shifted upwards to 780.00 ± 83.67 kHz when σswas increased to 0.10 S m-1. Dielectrophoretic and LCF spectra (translational speed of cells vs. electric field frequencies) of yeast suspensions during positive dielectrophoresis were analyzed in terms of the dielectric properties of the cell membrane and cytoplasm which reflect yeast cell viability and metabolic health status. The dielectrophoretic collection yield of cells using positive dielectrophoresis was reported on the monitor of sequential signal generator software to evaluate the number of living and dead cells through a real-time image processing analyzer. The spectra of both positive dielectrophoresis of the living and dead cells had distinguishable dielectric properties. The conductivity of the yeast cytoplasm (σc) of the dead cells was significantly less (≈ ≤ 0.05 S m-1) than that of the living yeast cells which typically had a cytoplasmic conductivity of ≈ 0.2 S m-1. This difference between viable and non-viable cells is sufficient for cell separation procedures. KEY POINTS: • Dielectrophoresis can be used to separate viable and non-viable yeast cells, • Cellular dielectric properties can be derived from the analysis of their dielectric spectra, • Cytoplasmic conductivity of viable cells is ≈ 0.2 S m-1 while that of non-viable cells ≈ ≤ 0.05 S m-1.

Evolution of the Tóxicos en Levadura 63 (TL63) gene family in plants and functional characterization of Arabidopsis thaliana TL63 under oxidative stress.

TL63 orthologs were angiosperm specific and had undergone motifs loss and gain, and increased purifying selection. AtTL63 was involved in the response of yeast and Arabidopsis plants to oxidative stress. The Tóxicos en Levadura (TL) family, a class of E3 ubiquitin ligases with typical RING-H2 type zinc finger structure, plays a pivotal role in mediating physiological processes and responding to stress in plants. However, the evolution and function of TL63 remain unclear. In this study, TL63 homologs were dated roughly back to the origin of land plants and confirmed to have subjected to the gain and loss of motifs and increased purifying selection. Phylogenetic analysis displayed that 279 TL63s could be divided into four main clades (Clade A-D). Notably, the ancestral tandem TL40/41 cluster contributed to the expansion of modern Brassicaceae TL40/41. The substitution rate tests revealed that the TL63 lineage was evidently different from other lineages. The codon usage index exhibited that monocotyledons preferred to use not A3s and T3s, but C3s, G3s, CAI, CBI and Fop. Sequence analysis showed that the TL63 homologs had conserved TM and GLD motifs and RING-H2 domain whose key amino acid residues accounted for the high average abundance. Particularly, Arabidopsis thaliana TL63 (AtTL63) was located in the nuclei, cell membranes and peroxisomes and expressed universally and significantly throughout A. thaliana development. Under H2O2 treatment, low or moderate expression of the AtTL63 held beneficial effects on the growth and viability of yeast cells and the mutation or overexpression of the AtTL63 positively affected the growth of A. thaliana plants. In brief, this study could supply useful insight into the evolution of the plant TL63s and the AtTL63 functions under oxidative stress.

Fast and Non-Invasive Evaluation of Yeast Viability in Fermentation Processes Using Raman Spectroscopy and Machine Learning

Fermentation processes used for producing alcoholic beverages such as beer, wine, and cider have a long history, having been developed early on across different civilizations. In most instances, yeast strains are used for fermentation processes, e.g., at breweries and wineries. Monitoring of yeast viability, cell count, and growth behavior is essential to ensure a controlled fermentation process. However, classical microbiological techniques to monitor fermentation process parameters are time-consuming and require sampling, along with the risk of contamination. Nowadays, industries are moving toward automation and digitalization. This necessitates state-of-the-art process analytical technologies to ensure an efficient and controlled process to obtain high-quality product outputs. Hence, there is a strong need for a fast, non-invasive, and generally applicable method to evaluate the viability of yeast cells during fermentation to warrant the standardization and purity of produced products in industrial applications. The aim of our study is to discriminate between viable and non-viable yeast in various culture media using Raman spectroscopy (RS) followed by data analysis with machine learning (ML) tools. These techniques allow for rapid, non-invasive analysis addressing the limitations of traditional methods. The present work primarily focuses on the evaluation of RS combined with predictive ML models in a non-real-time setting. Our goal is to adapt these techniques for future application in real-time monitoring and determination of yeast viability in biotechnological processes. We demonstrate that RS, in combination with ML, is a promising tool for non-invasive inline monitoring of fermentation processes.

Binding of human Cdc123 to eIF2γ

Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly.

Antifungal and antibacterial activities of Cannabis sativa L. resins.

Ethnopharmacological relevanceCannabis sativa L. (Cannabaceae) is a plant native to Eastern Asia spread throughout the world because of its medicinal properties. Despite being used for thousands of years as a palliative therapeutic agent for many pathologies, in many countries research on its effects and properties could only be carried out in recent years, after its legalization. Aims of the studyIncreasing resistance to traditional antimicrobial agents demands finding new strategies to fight against microbial infections in medical therapy and agricultural activities. Upon legalization in many countries, Cannabis sativa is gaining attention as a new source of active components, and the evidence for new applications of these compounds is constantly increasing. MethodsExtracts from five different varieties ofCannabis sativa were performed and their cannabinoids and terpenes profiles were determined by liquid and gas chromatography. Antimicrobial and antifungal activities against Gram (+) and Gram (−) bacteria, yeast and phytopathogen fungus were measured. To analyze a possible action mechanism, cell viability of bacteria and yeast was assessed by propidium iodide stain. ResultsCannabis varieties were grouped into chemotype I and II as a consequence of their cannabidiol (CBD) or tetrahydrocannabinol (THC) content. The terpenes profile was different in quantity and quality among varieties, with (−)b-pinene, b-myrcene, p-cymene and b-caryophyllene being present in all plants. All cannabis varieties were effective to different degree against Gram (+) and Gram (−) bacteria as well as on spore germination and vegetative development of phytopathogenic fungi. These effects were not correlated to the content of major cannabinoids such as CBD or THC, but with the presence of a complex terpenes profile. The effectiveness of the extracts allowed to reduce the necessary doses of a widely used commercial antifungal to prevent the development of fungal spores. ConclusionAll the extracts of the analysed cannabis varieties showed antibacterial and antifungal activities. In addition, plants belonging to the same chemotype showed different antimicrobial activity, demonstrating that the classification of cannabis strains based solely on THC and CBD content is not sufficient to justify their biological activities and that other compounds present in the extracts are involved in their action against pathogens. Cannabis extracts act in synergy with chemical fungicides, allowing to reduce its doses.