Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance

Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated tEpe1 which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non- cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell-integrity MAP kinase stress signalling pathway, mutations in which alter resistance. Thus, environmental changes provoke a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered.

Integrity of Sperm Cell Membrane in the Semen of Crossbred and Purebred Boars during Storage at 17 °C: Heterosis Effects

The aim of the study was to assess changes in the integrity of sperm cell membranes during the storage of semen collected from Duroc × Pietrain crossbred boars and purebred boars of the component breeds. To compare the cell membrane integrity of sperm heads in crossbred and purebred boars, heterosis effects were estimated. The study was conducted on 48 ejaculates collected from Duroc × Pietrain crossbred boars and from purebred Duroc and Pietrain boars used for artificial insemination. Microscope slides were prepared from each ejaculate for the evaluation of the cell membrane integrity of the sperm, at 1, 24, 48, 72, and 96 h after collection of the ejaculate. Diluted ejaculates were stored at 17 °C. Sperm membrane integrity was analysed by two methods: SYBR-14/PI and eosin–nigrosin. Our results showed that the cell membrane integrity of sperm heads changed with storage time, but the extent of the changes varied depending on the genetic group of boars. The semen of Duroc × Pietrain crossbreds was clearly seen to be less sensitive to storage conditions than that of boars of the parent breeds, which was confirmed by the calculated heterosis effects. The percentage of sperm with an intact cell membrane was higher in crossbred boars than in purebred boars (p ≤ 0.05). In addition, significantly fewer moribund sperm spermatozoa and spermatozoa with a damaged cell membrane were observed in crossbred boars (p ≤ 0.05). In the semen of purebred Duroc and Pietrain boars, the cell membrane integrity of the sperm should be assessed more often during storage than in the semen of Duroc × Pietrain crossbred boars. This study provides valuable information for the development and implementation of semen quality monitoring in crossbred boars and boars of the parent breeds during storage at 17 °C with respect to the cell membrane structure of sperm heads. The evaluation methods used effectively identify damage to the cell membranes of the sperm during semen storage.

A Novel Approach to the Viability Determination of Mycobacterium avium subsp. paratuberculosis Using Platinum Compounds in Combination With Quantitative PCR

Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.

Rotating Magnetic Field Increases β-Lactam Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have developed resistance to most β-lactam antibiotics and have become a global health issue. In this work, we analyzed the impact of a rotating magnetic field (RMF) of well-defined and strictly controlled characteristics coupled with β-lactam antibiotics against a total of 28 methicillin-resistant and sensitive S. aureus strains. The results indicate that the application of RMF combined with β-lactam antibiotics correlated with favorable changes in growth inhibition zones or in minimal inhibitory concentrations of the antibiotics compared to controls unexposed to RMF. Fluorescence microscopy indicated a drop in the relative number of cells with intact cell walls after exposure to RMF. These findings were additionally supported by the use of SEM and TEM microscopy, which revealed morphological alterations of RMF-exposed cells manifested by change of shape, drop in cell wall density and cytoplasm condensation. The obtained results indicate that the originally limited impact of β-lactam antibiotics in MRSA is boosted by the disturbances caused by RMF in the bacterial cell walls. Taking into account the high clinical need for new therapeutic options, effective against MRSA, the data presented in this study have high developmental potential and could serve as a basis for new treatment options for MRSA infections.

The periodic table of photosynthetic purple non-sulfur bacteria: intact cell-metal ions interactions

Photosynthetic purple non-sulfur bacteria (PNB) have been widely utilized as model organisms to study bacterial photosynthesis. More recently, the remarkable resistance of these microorganisms to several metals ions called particular interest. As a result, several research efforts were directed toward clarifying the interactions of metal ions with PNB. The mechanisms of metal ions active uptake and bioabsorption have been studied in detail, unveiling that PNB enable harvesting and removing various toxic ions, thus fostering applications in environmental remediation. Herein, we present the most important achievements in the understanding of intact cell-metal ions interactions and the approaches utilized to study such processes. Following, the application of PNB-metal ions interactions toward metal removal from contaminated environments is presented. Finally, the possible coupling of PNB with abiotic electrodes to obtain biohybrid electrochemical systems is proposed as a sustainable pathway to tune and enhance metal removal and monitoring. Graphic abstract

Effect of Metabolism of Carbohydrate in Mammalian Tissues and Tumors on The Level of Enzymes Involved in Direct Oxidative Pathway

Introduction: Literature review has revealed that the distribution of the enzymes of 6-phospo-gluconate dehydrogenase activities of some acetone derived tissues in animal tissue have so far not been investigated systematically. This leaves a gap for further investigation to explore the subject matter deeply. Method: Barium salts of D-Glucose 6-phosphate (0 6-P), 6-phosphogluconate (6-PO) and D-ribose 5-phosphate (R 5-P) are available and were used in our study. (TNP) triphosphopyridine was prepared and analyzed; its composition was 75% TNP without (DNP) diphosphopyridine nucleotide. Ice-cold isotone KCL (0-15M-KCL with 8ml, 0-02M-KHCO3) was disintegrated in 09 parts. It is done in a potter glass homogenizer or in a Nelco homogenizer. This is followed by centrifuging and dialysis of the supernatants. Heparinized blood 10ml was used for erythrocyte hemolysis, which was diluted with 10ml of water. 01 part of haemolysate was treated with 9 parts of isotonic KCI. Spectroscope is used to determine the dehydrogenase activity of dialyzed tissue. The method followed was of Glock and McLean. Study Design: Quantitative, cross sectional study. Settings: Institute of Biochemistry, Gulab Devi Educational Complex, Lahore Duration: 01 Year i.e. 1st July 2020 to 30th June 2021. Results: Enzymes activities of 6-PG dehydrogenase and Gluco-6- phospo dehydrogenase mentioned in table 1&2 in normal mammalian tissue and mammary glands. The results obtained on the tumour cells are given in table 3. These Values are within the limits in normal tissues whereas it becomes on higher side in lymphomas and sarcomas. Conclusion: This study shows some limitation that the maximum enzymic activities are determined, whereas in the intact cell other regulatory factors probably limit or control the activity of this pathway. Keywords: Gluco-6- phospodehydrogenase, 6-PG dehydrogenase, oxidative pathway, Ribose 5-Pentose, mammalian tissue

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

Plasma membranes contain diverse nanoscale assemblies of lipid and protein domains. Specific localization of lipids and proteins in these domains is often essential for membrane function and integrity. Due to the nanoscale size and dynamic nature of membrane domains, identification of molecules residing in domains either is not possible with modern imaging techniques or requires advanced methods with high spatiotemporal resolution. Such methods need expensive equipment making these approaches inaccessible and thus difficult to implement at large scale. Here, we present a novel membrane fluidizer-induced clustering (MFIC) approach to identify the phase-preference of molecules in intact cell membranes. Experiments in phase-separated bilayers and live cells on molecules with known phase preference demonstrate that heptanol hyperfluidizes the membrane and stabilizes phase separation in cell membranes. The domain stabilization results in a transition of nano- to micron-sized clusters of associated molecules and allows identification of molecules localized in domains using routine microscopy techniques. This assay can be carried out on both genetically and extrinsically labelled molecules in live cell membranes, does not require any invasive sample preparation and can be carried out in 10-15 minutes. This inexpensive and easy to implement assay can be conducted at large-scale and will allow easy identification of molecules partitioning into domains.

LON DELETION IMPAIRS PERSISTER CELL RESUSCITATION IN ESCHERICHIA COLI

Bacterial persisters are non-growing cells that are highly tolerant to bactericidal antibiotics. However, this tolerance is reversible and not mediated by heritable genetic changes. Lon, an ATP-dependent protease, has repeatedly been shown to play a critical role in fluoroquinolone persistence. Although lon deletion (Δlon) is thought to kill persister cells via accumulation of the cell division inhibitor protein SulA, the exact mechanism underlying this phenomenon has yet to be elucidated. Here, we show that Lon is an important regulatory protein for the resuscitation of the fluoroquinolone persisters in Escherichia coli, and lon deletion impairs the ability of persister cells to form colonies during recovery, without killing these cells, through a sulA- and ftsZ-dependent mechanism. Notably, this observed non-culturable state of antibiotic-tolerant Δlon cells is transient, as environmental conditions, such as starvation, can restore their culturability. Our data further indicate that starvation-induced SulA degradation or expression of Lon during recovery facilitates Z-ring formation in Δlon persisters. Calculating the ratio of the cell length (L in µm) to the number of Z-rings (Z) for each ofloxacin-treated intact cell analyzed has revealed a strong correlation between persister resuscitation and calculated L/Z values, which represents a potential biomarker for Δlon persisters that are transitioning to the normal cell state under the conditions studied here.

Optimization of polycaprolactone - based nanofiber matrices for the cultivation of corneal endothelial cells

Posterior lamellar transplantation of the eye’ s cornea (DSAEK, DMEK) currently is the gold standard for treating patients with corneal endothelial cell and back surface pathologies resulting in functional impairment. An artificial biomimetic graft carrying human corneal endothelium could minimize the dependency on human donor corneas giving access to this vision-restoring surgery to large numbers of patients, thus reducing current long waiting lists. In this study, four groups of electrospun nanofibrous scaffolds were compared: polycaprolactone (PCL), PCL/collagen, PCL/gelatin and PCL/chitosan. Each of the scaffolds were tissue-engineered with human corneal endothelial cells (HCEC-B4G12) and analyzed with regard to their potential application as artificial posterior lamellar grafts. Staining with ZO-1 and Na+/K+-ATPase antibodies revealed intact cell functionalities. It could be shown, that blending leads to decreasing contact angle, whereby a heterogeneous blend morphology could be revealed. Scaffold cytocompatibility could be confirmed for all groups via live/dead staining, whereby a significant higher cell viability could be observed for the collagen and gelatine blended matrices with 97 ± 3% and 98 ± 2% living cells respectively. TEM images show the superficial anchoring of the HCECs onto the scaffolds. This work emphasizes the benefit of blended PCL nanofibrous scaffolds for corneal endothelial keratoplasty.