A Simple and Efficient Mechanical Cell Disruption Method Using Glass Beads to Extract β-Glucans from Spent Brewer’s Yeast

β-glucan extraction from spent brewer’s yeast is a long process that starts with the lysis of yeast cells, this step lasting up to 36 h and can be disadvantageous when working on a small scale. In this study, a rapid cell rupture method was selected for the lysis of spent brewer’s yeast to obtain β-glucans. Optimal parameters were determined for the lysis of a cellular suspension of spent brewer’s yeast by vortexing with glass beads. Thus, parameters such as the number of 10 min vortex cycles from 1 to 3, the concentration of cell suspension (5, 10, and 15%), and the ratio of yeast/glass beads (1:1, 1:2, and 1:3) were varied in a Box-Behnken design. A cell lysis mechanism using glass beads allows the cell to rupture and permits the removal of intracellular content. An increase in yeast suspension concentration decreased the disruption efficiency, while a proportional increase was observed with the yeast/glass beads ratio and the increasing number of vortexing cycles. The optimal parameters for cell lysis were found to be a cell suspension concentration of 5%, a ratio of yeast/glass beads of 1:2, and a vortexing cycle of 3, with a disruption efficiency of 99.8%. The β-glucan fraction extracted from the optimal sample showed characteristic absorption bands at 1370.77 and 1153.92 cm−1, the content of β-glucan being 78.53%.

Cell lysis analysis for respiratory viruses through simulation modeling

An ordinary system of differential equations leading to a simulation model is propose as methodological approach to analysis the incidence of infectious-contagious diseases, in this case using SARS-CoV-2 virus as pathogenic model. The dynamics of the model are drive by the interaction between susceptible cells contemplating respiratory epithelial cells and viral infection mediated by two types of lysis response. To perform the simulations, values of some variables and parameters were selected from referenced sources, considering that previous reports suggested that the viral load in the lower respiratory tract might reach its peak in the second week after the beginning of disease symptoms. The scenarios described in the simulations evidence the performance of the cell lysis response from susceptible cells that have been infected. The recommend model shows that an excess response from both the original virus and the mutated virus leads to an increase in the approximate time to control viral infection within the organism.

Plasma membrane perforation by GSDME during apoptosis-driven secondary necrosis

Secondary necrosis has long been perceived as an uncontrolled process resulting in total lysis of the apoptotic cell. Recently, it was shown that progression of apoptosis to secondary necrosis is regulated by Gasdermin E (GSDME), which requires activation by caspase-3. Although the contribution of GSDME in this context has been attributed to its pore-forming capacity, little is known about the kinetics and size characteristics of this. Here we report on the membrane permeabilizing features of GSDME by monitoring the influx and efflux of dextrans of different sizes into/from anti-Fas-treated L929sAhFas cells undergoing apoptosis-driven secondary necrosis. We found that GSDME accelerates cell lysis measured by SYTOX Blue staining but does not affect the exposure of phosphatidylserine on the plasma membrane. Furthermore, loss of GSDME expression clearly hampered the influx of fluorescently labeled dextrans while the efflux happened independently of the presence or absence of GSDME expression. Importantly, both in- and efflux of dextrans were dependent on their molecular weight. Altogether, our results demonstrate that GSDME regulates the passage of compounds together with other plasma membrane destabilizing subroutines.

Novel Combinations of Human Immunomodulatory mAbs Lacking Cardiotoxic Effects for Therapy of TNBC

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by a higher mortality rate among breast cancer subtypes. Poly(ADP-ribose) polymerase (PARP) inhibitors are used in clinics to treat a subgroup of TNBC patients, but other targeted therapies are urgently needed. Programmed death-ligand 1 (PD-L1), involved in tumor immune escape, was recently identified as a target for TNBC; accordingly, the anti-PD-L1 monoclonal antibody (mAb), atezolizumab, has been approved by FDA in combination with Paclitaxel for the therapy of metastatic TNBC. Here, we tested novel combinations of fully human immunomodulatory mAbs, including anti-PD-L1 mAbs generated in our laboratory and atezolizumab, on TNBC and other tumor cell lines. We evaluated their anti-tumor efficacy when used as single agents or in combinatorial treatments with anti-CTLA-4 mAbs in in vitro co-cultures of hPBMCs with tumor cells, by measuring tumor cell lysis and IL-2 and IFNγ cytokines secretion by lymphocytes. In parallel, by using co-cultures of hPBMCs and cardiomyocytes, we analyzed the potential cardiotoxic adverse side effects of the same antibody treatments by measuring the cardiac cell lysis and the secretion of pro-inflammatory cytokines. We identified novel combinations of immunomodulatory mAbs endowed with more potent anti-cancer activity on TNBC and lower cardiotoxic side effects than the combination of atezolizumab and ipilimumab.

Curcumin activation of a bacterial mechanosensitive channel underlies its membrane permeability and adjuvant properties

Curcumin, a natural compound isolated from the rhizome of turmeric, has been shown to have antibacterial properties. It has several physiological effects on bacteria including an apoptosis-like response involving RecA, membrane permeabilization, inhibiting septation, and it can also work synergistically with other antibiotics. The mechanism by which curcumin permeabilizes the bacterial membrane has been unclear. Most bacterial species contain a Mechanosensitive channel of large conductance, MscL, which serves the function of a biological emergency release valve; these large-pore channels open in response to membrane tension from osmotic shifts and, to avoid cell lysis, allow the release of solutes from the cytoplasm. Here we show that the MscL channel underlies the membrane permeabilization by curcumin as well as its synergistic properties with other antibiotics, by allowing access of antibiotics to the cytoplasm; MscL also appears to have an inhibitory role in septation, which is enhanced when activated by curcumin.

Echinocandin Drugs Induce Differential Effects in Cytokinesis Progression and Cell Integrity

Fission yeast contains three essential β(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of bgs1+ and bgs3+. Thus, Bgs1 and Bgs3 functions seem to be unaffected by those GS inhibitors. To learn more about echinocandins’ mechanism of action and resistance, cytokinesis progression and cell death were examined by time-lapse fluorescence microscopy in WT and pbr1-8 cells at the start of treatment with sublethal and lethal concentrations of anidulafungin, caspofungin, and micafungin. In WT, sublethal concentrations of the three drugs caused abundant cell death that was either suppressed (anidulafungin and micafungin) or greatly reduced (caspofungin) in pbr1-8 cells. Interestingly, the lethal concentrations induced differential phenotypes depending on the echinocandin used. Anidulafungin and caspofungin were mostly fungistatic, heavily impairing cytokinesis progression in both WT and pbr1-8. As with sublethal concentrations, lethal concentrations of micafungin were primarily fungicidal in WT cells, causing cell lysis without impairing cytokinesis. The lytic phenotype was suppressed again in pbr1-8 cells. Our results suggest that micafungin always exerts its fungicidal effect by solely inhibiting Bgs4. In contrast, lethal concentrations of anidulafungin and caspofungin cause an early cytokinesis arrest, probably by the combined inhibition of several GSs.

Investigating the Mechanism Behind the Release of Microcystins in Freshwater Cyanobacteria

<p>The frequency and distribution of toxic cyanobacterial blooms are increasing globally, creating the need for a better understanding of the processes involved in toxic secondary metabolite production. Microcystins (MCs) are potent hepatotoxins produced by a wide range of bloom-forming cyanobacteria genera such as Microcystis and Planktothrix. Although the release of MCs to the extracellular environment has long been considered a by-product of cell lysis and death, several studies suggest the presence of a mechanism that actively transports these toxins outside the cell membrane. The aim of the present study was to find evidence for a link between cell lysis and concentrations of extracellular MCs. A dual-fluorescence cell viability assay using the nucleic acid stain SYTOX Green was optimised for use on Microcystis and Planktothrix. A SYTOX Green concentration of 1 µM, and an incubation time of 30 minutes, yielded a bright and even fluorescent signal that readily identified lysed cells. The improved staining technique, in conjunction with liquid chromatography-mass spectrometry analyses, was employed in a culturing experiment to track the transfer of MCs to the extracellular environment in relation to the amount of cell lysis. For Microcystis, there was a strong and significant positive relationship between cell lysis and the concentration of extracellular MC. When the extracellular MC was predicted according to cell lysis levels and the MC content per cell, lysed cells were a major contributor of MCs to the extracellular environment, although the model overestimated the concentrations. Relationships for Planktothrix were significant but weaker, possibly due to reduced accuracy in the cell enumeration step, which would have altered the calculated MC content per cell. Whilst these findings support the hypothesis that cell lysis is the main contributor of extracellular MCs, the results do not exclude a role of MCs as signalling molecules. The recent finding that programmed cell death may occur in Microcystis under various environmental conditions may explain the commonly observed increase in extracellular MCs. Understanding the mechanisms involved in the transfer of MCs to the extracellular environment will provide further clarification on the function of these secondary metabolites and lead to the improvement of water quality management strategies.</p>

Investigating the Mechanism Behind the Release of Microcystins in Freshwater Cyanobacteria

<p>The frequency and distribution of toxic cyanobacterial blooms are increasing globally, creating the need for a better understanding of the processes involved in toxic secondary metabolite production. Microcystins (MCs) are potent hepatotoxins produced by a wide range of bloom-forming cyanobacteria genera such as Microcystis and Planktothrix. Although the release of MCs to the extracellular environment has long been considered a by-product of cell lysis and death, several studies suggest the presence of a mechanism that actively transports these toxins outside the cell membrane. The aim of the present study was to find evidence for a link between cell lysis and concentrations of extracellular MCs. A dual-fluorescence cell viability assay using the nucleic acid stain SYTOX Green was optimised for use on Microcystis and Planktothrix. A SYTOX Green concentration of 1 µM, and an incubation time of 30 minutes, yielded a bright and even fluorescent signal that readily identified lysed cells. The improved staining technique, in conjunction with liquid chromatography-mass spectrometry analyses, was employed in a culturing experiment to track the transfer of MCs to the extracellular environment in relation to the amount of cell lysis. For Microcystis, there was a strong and significant positive relationship between cell lysis and the concentration of extracellular MC. When the extracellular MC was predicted according to cell lysis levels and the MC content per cell, lysed cells were a major contributor of MCs to the extracellular environment, although the model overestimated the concentrations. Relationships for Planktothrix were significant but weaker, possibly due to reduced accuracy in the cell enumeration step, which would have altered the calculated MC content per cell. Whilst these findings support the hypothesis that cell lysis is the main contributor of extracellular MCs, the results do not exclude a role of MCs as signalling molecules. The recent finding that programmed cell death may occur in Microcystis under various environmental conditions may explain the commonly observed increase in extracellular MCs. Understanding the mechanisms involved in the transfer of MCs to the extracellular environment will provide further clarification on the function of these secondary metabolites and lead to the improvement of water quality management strategies.</p>

Picoliter liquid handling at gas/liquid interface by surface and geometry control in a micro-nanofluidic device

In micro- and nanofluidic devices, highly precise fluidic control is essential. Conventional mechanical valves in microchannels and nanochannels have size limitations, whereas hydrophobic (Laplace) valves are generally difficult to use for low-surface-tension liquids. In the present study, we developed a method for handling picoliter volumes of low-surface-tension liquids in a micro-nanofluidic device. The proposed Laplace valve is based on the pinning effect. A fused silica micro-nanofluidic device that includes a picoliter chamber whose geometry was designed to induce capillary pinning was designed and fabricated. The measured Laplace pressure of a lysis buffer (surfactant) was consistent with the calculated pressure, indicating successful fabrication and hydrophobic surface modification. The working principle of the Laplace valve was verified. The Laplace valve maintained the lysis buffer at the gas/liquid interface for 60 min, which is sufficiently long for cell lysis operations. Finally, replacement of liquids in the picoliter chamber using the valve was demonstrated. The proposed method will contribute to basic technologies for fluidic control in micro- and nanofluidic devices, and the proposed Laplace valve can be used for low-surface-tension liquids. In addition, the developed valve and picoliter chamber can be utilized for the interface in single-cell lysis, which will facilitate the development of single-cell analysis devices.