Cell Membrane Lysis Research Articles

Two New Lytic Bacteriophages of the Myoviridae Family Against Carbapenem-Resistant Acinetobacter baumannii.

Two lytic bacteriophages, WCHABP1 and WCHABP12, were recovered from hospital sewage and were able to infect 9 and 12 out of 18 carbapenem-resistant Acinetobacter baumannii clinical strains, which belonged to different clones. Electron microscopy scan showed that both bacteriophages had the similar morphology as those of the Myoviridae family. Whole genomic sequencing revealed 45.4- or 45.8-kb genome with a 37.6% GC content for WCHABP1 and WCHABP12, both of which showed significant DNA sequence similarity with bacteriophages of the Ap22virus genus within the Myoviridae family. Taxonomic analysis was therefore performed following the proposal approved by the International Committee on Taxonomy of Viruses, which confirmed that WCHABP1 and WCHABP12 represented two new species of the Ap22virus genus. No tRNAs but 88 and 89 open reading frames (ORFs) were predicted for the two bacteriophages, among which 22 and 21 had known function and encoded proteins for morphogenesis, packaging, lysis, and nucleiotide metabolism. The C-terminal amino acids of the large unit of fiber tail proteins varied between the bacteriophages, which may explain their different host ranges. For most lytic bacteriophages, a set of holin and endolysin are required for lysis. However, no known holin-encoding genes were identified in WCHABP1 and WCHABP12, suggesting that they may use alternative, yet-to-be-identified, novel holins for host cell membrane lysis. To test the efficacy of the bacteriophages in protecting against A. baumannii infection, a Galleria mellonella larva model was used. Only <20% G. mellonella larvae survived at 96 h after being infected by carbapenem-resistant A. baumannii strains, from which the two bacteriophages were recovered. With the administration of WCHABP1 and WCHABP12, the survival of larvae increased to 75%, while the treatment of polymyxin B only slightly increased the survival rate to 25%. The isolation of two new lytic bacteriophages in this study could expand our sight on Acinetobacter bacteriophages and may offer new potential therapeutic alternatives against A. baumannii.

Broad-Spectrum Antibacterial Activity of Proteolytically Stable Self-Assembled αγ-Hybrid Peptide Gels.

Bacterial infections pose a serious threat to mankind, and there is immense interest in the design and development of self-assembled peptide gels using ultrashort peptides for antibacterial applications. The peptide gels containing natural amino acids suffer from poor stability against proteolytic enzymes. Therefore, there is a need to design and develop peptide gels with improved stability against proteolytic enzymes. In the present work, we report the synthesis and characterization of α/γ hybrid peptides Boc-D-Phe-γ4-L-Phe-PEA (NH007) and Boc-L-Phe-γ4-L-Phe-PEA (NH009) to improve the proteolytic stability. Both of the dipeptides were found to self-assemble into gels in aqueous DMSO (3-5% w/v), and the self-assembly process was studied using FTIR and CD, which indicated antiparallel β-sheet formation with random coils in NH007 gels and random or unordered conformation in NH009. The rheological studies indicated viscoelastic characteristics for both gels; the storage modulus ( G') for NH007 and NH009 gels (3% w/v) was estimated as 0.2 and 0.5 MPa, higher than the loss modulus ( G''). Also, both gels demonstrated self-healing characteristics for six consecutive cycles when subjected to varying strains of 0.1 and 30% (200 s each). The peptide gels were incubated with a mocktail of proteolytic enzymes, proteinase K, pepsin, and chymotrypsin, and stability was monitored using RP HPLC. Up to 23 and 40% degradation was observed for NH007 (3%, w/v) in 24 and 36 h, and 77 and 94% degradation was observed for NH009 (3%, w/v), within the same period. Thus α/γ hybrid peptide gels containing D-Phe exhibited higher stability than gels fabricated using L-Phe. The use of D-residue in α/γ hybrid peptide significantly enhanced the stability of peptides against proteolytic enzymes, as the stability data reported in this work are possibly the best in class. Both peptide gels exhibited broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The Pseudomonas aeruginosa and Staphylococcus aureus, in particular, are known to develop resistance. The NH007 (3%, w/v) demonstrated 65% inhibition, whereas NH009 (3%, w/v) showed 78% inhibition, with potent activity against Pseudomonas aeruginosa. Mechanistic studies, using SEM, HR-TEM, and bacterial live-dead assay, indicated entrapment of bacteria in gel networks, followed by interaction with cell membrane components and lysis. Cell viability (MTT assay) and toxicity (LDH assay) studies showed that both gels are not toxic to NIH 3T3 mouse embryonic fibroblast cells (mammalian). MTT assay showed >85% cell viability, and LDH assay exhibited not more than 15% cytotoxicity, even at higher concentrations (5%, w/v) and prolonged exposures (48 h). Overall, studies indicate the potential application of gels developed from the α/γ hybrid peptides in preventing biomaterial-related infections.

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

P066 Structural aspects of HLA class I epitopes reacting with human alloantiserum

Aim Interaction between complementarity determining regions (CDR) of the antibody (Ab) and HLA (contact surface area of 700–900 A2) can be detected by mean fluorescence intensity (MFI) or lysis of cell membrane by complement dependent cytotoxicity (CDC). These biological functions of Ab depend on amino acid residue (AAR) composition of functional and structural epitopes (Ep) of the antigen. The aim of this study was to analyze Ep structure of HLA in order to interpret reactivity of Abs in the serum of kidney recipient (KR). Methods Ab analysis (IgG) and complement binding activity (C1q test) of alloantibodies were analyzed using solid phase single antigen (One Lambda, Inc.) (positive cut off MFI = 280–500) and cell based CDCx3 washes assays. Ep analysis was conducted by using HLAMatchmaker computer algorithm (MFI = 1000 was used as a positive cut off). Results Various combinations of four (63EE, 82LR, 127K, and 113YH) eplet-defined Eps were detected in HLAs Abs in KR serum reacted with, when SAA was applied. Consensus of AAR within 15 A radius of aforementioned eplets comprised sixteen positions. The results of reactivity of specific eplet carrying alleles in three assays are presented in Fig. 1. Our findings indicate that Ab reactivity is associated with AAR composition of structural Eps involved in contact with CDR of IgG. Binding to HLA likely depends on AARs at positions 44, 152, 156, and 158, whereas C1q reactivity is affected by AARs at 9H/24T and 163E/167S positions, and CDC reactivity is abolished by tryptophan (W) at 95 position. Conclusions Biological function of anti-HLA Ab requires not only the specific recognition of eplets but also depends on the interactions of their CDRs with structural Eps within the contact surface area. C1q + test does not always predict a positive CDC assay. The results of this study represent a new insight into organ allocation, immunomodulation therapy, and post-transplant Ab monitoring in solid organ recipients. Download : Download high-res image (603KB) Download : Download full-size image

Biodegradable cationic poly(carbonates): Effect of varying side chain hydrophobicity on key aspects of gene transfection.

Owing to their ease of synthesis and well-controlled polymerization, biodegradable cationic poly(carbonates) have emerged as a highly promising class of biomaterials for gene delivery. The hydrophobicity of side chains in cationic polymers plays an important but often underappreciated role in influencing key aspects of gene transfection. In our efforts to improve gene transfection and understand structure-activity relationships, we synthesized a series of cationic polymers bearing a common poly(carbonate) backbone, and with side chains containing various hydrophobic spacers (propyl, hexyl, 4-methyl benzyl or nonyl) before the cationic moiety. A moderate degree of hydrophobicity was optimal as the cationic poly(carbonate) with hexyl side chains mediated high gene transfection efficiencies while causing low cytotoxicities.

Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions

Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1-secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1-dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1-mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1-mediated lysis.

Replacement of fishmeal with processed meal from knife fish Chitala ornata in diets of juvenile Nile tilapia Oreochromis niloticus

A 60-day feeding trial was conducted to assess the effects of processed meal from knife fish Chitala ornata (KFM) as fishmeal replacement in diets of Nile tilapia Oreochromis niloticus juveniles. Five iso-nitrogenous (36.4% in dry matter) and isolipidic diets (8.6% in dry matter) with 0 (D1), 25 (D2), 50 (D3), 75 (D4) and 100% (D5) KFM inclusions were prepared. With a stocking density of 15 fish (0.59±0.01g) per tank, tilapia juveniles were distributed randomly in fifteen 30-L tanks. Results indicate a significant increase (P<0.05) in the percent average weight gain, specific growth rate, and feed intake with increasing KFM inclusion up to the level of 75%. There were no adverse effects observed in both blood profile and carcass composition of the tilapia. Hepatosomatic index of fish significantly increased (P<0.05) when KFM was included into the diet of tilapia, compared to those fed D1. Viscerosomatic indices were not significantly different (P>0.05) among treatments. Results of hepatic histopathology showed absence of tumors, lesions and parenchymal inflammation in all treatments. However, mild cell membrane lysis and mild and mild to moderate apoptosis were evident in liver samples. Based on the results, KFM can partially and completely replace dietary protein from fishmeal. Moreover, D4 (75% KFM) is considered the optimal KFM replacement level for Nile tilapia juveniles.

Microfluidic Platform for Parallel Single Cell Analysis for Diagnostic Applications.

Cell populations are heterogeneous: they can comprise different cell types or even cells at different stages of the cell cycle and/or of biological processes. Furthermore, molecular processes taking place in cells are stochastic in nature. Therefore, cellular analysis must be brought down to the single cell level to get useful insight into biological processes, and to access essential molecular information that would be lost when using a cell population analysis approach. Furthermore, to fully characterize a cell population, ideally, information both at the single cell level and on the whole cell population is required, which calls for analyzing each individual cell in a population in a parallel manner. This single cell level analysis approach is particularly important for diagnostic applications to unravel molecular perturbations at the onset of a disease, to identify biomarkers, and for personalized medicine, not only because of the heterogeneity of the cell sample, but also due to the availability of a reduced amount of cells, or even unique cells. This chapter presents a versatile platform meant for the parallel analysis of individual cells, with a particular focus on diagnostic applications and the analysis of cancer cells. We first describe one essential step of this parallel single cell analysis protocol, which is the trapping of individual cells in dedicated structures. Following this, we report different steps of a whole analytical process, including on-chip cell staining and imaging, cell membrane permeabilization and/or lysis using either chemical or physical means, and retrieval of the cell molecular content in dedicated channels for further analysis. This series of experiments illustrates the versatility of the herein-presented platform and its suitability for various analysis schemes and different analytical purposes.

Ischemia/Reperfusion.

Ischemic disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are the most common causes of debilitating disease and death in westernized cultures. The extent of tissue injury relates directly to the extent of blood flow reduction and to the length of the ischemic period, which influence the levels to which cellular ATP and intracellular pH are reduced. By impairing ATPase-dependent ion transport, ischemia causes intracellular and mitochondrial calcium levels to increase (calcium overload). Cell volume regulatory mechanisms are also disrupted by the lack of ATP, which can induce lysis of organelle and plasma membranes. Reperfusion, although required to salvage oxygen-starved tissues, produces paradoxical tissue responses that fuel the production of reactive oxygen species (oxygen paradox), sequestration of proinflammatory immunocytes in ischemic tissues, endoplasmic reticulum stress, and development of postischemic capillary no-reflow, which amplify tissue injury. These pathologic events culminate in opening of mitochondrial permeability transition pores as a common end-effector of ischemia/reperfusion (I/R)-induced cell lysis and death. Emerging concepts include the influence of the intestinal microbiome, fetal programming, epigenetic changes, and microparticles in the pathogenesis of I/R. The overall goal of this review is to describe these and other mechanisms that contribute to I/R injury. Because so many different deleterious events participate in I/R, it is clear that therapeutic approaches will be effective only when multiple pathologic processes are targeted. In addition, the translational significance of I/R research will be enhanced by much wider use of animal models that incorporate the complicating effects of risk factors for cardiovascular disease. © 2017 American Physiological Society. Compr Physiol 7:113-170, 2017.

Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array.

A re-examination of the role of the acute phase protein response in innate cancer defence

Anti-cancer host defense mechanisms are traditionally considered to consist of tumor suppressor genes and immune surveillance by cells of the innate and adaptive immune systems. However, there is mounting evidence that components of the acute phase protein response (APPR), and, in particular, certain cationic host defense peptides (HDPs), also contribute to anti-cancer host defense. In a number of in vitro studies, certain HDPs have been shown to be cytotoxic to tumor cells either directly through cancer cell membrane destabilization and lysis or through the initiation of apoptosis in the cancer cell. In addition, many cancer cells elaborate the pro-inflammatory cytokine interleukin-6, which in turn produces an APPR that involves the release of HDPs. It is therefore possible that the release of pro-inflammatory cytokines by cancer cells initiates a poorly understood anti-tumor response by the host that involves HDP induction. We hypothesize that the APPR may form an important anti-cancer host defense response. This may be an important consideration in light of cancer treatments designed to decrease systemic inflammation. Blunting of the anti-cancer effect of the APPR may also contribute to the increased cancer rates seen in chronic immunosuppressive states.

Apoptosis and morphological alterations after UVA irradiation in red blood cells of p53 deficient Japanese medaka (Oryzias latipes)

Morphological alterations in red blood cells were described as hematological bioindicators of UVA exposure to investigate the sensitivity to UVA in wild type Japanese medaka (Oryzias latipes) and a p53 deficient mutant. The fewer abnormal red blood cells were observed in the p53 mutant fish under the control conditions. After exposure to different doses of UVA radiation (15min, 30min and 60min/day for 3days), cellular and nuclear alterations in red blood cells were analyzed in the UVA exposed fish compared with non-exposed controls and those alterations included acanthocytes, cell membrane lysis, swollen cells, teardrop-like cell, hemolyzed cells and sickle cells. Those alterations were increased after the UVA exposure both in wild type and the p53 deficient fish. Moreover, apoptosis analyzed by acridine orange assay showed increased number of apoptosis in red blood cells at the higher UVA exposure dose. No micronuclei but nuclear abnormalities as eccentric nucleus, nuclear budding, deformed nucleus, and bilobed nucleus were observed in each group. These results suggested that UVA exposure induced both p53 dependent and independent apoptosis and morphological alterations in red blood cells but less sensitive to UVA than Wild type in medaka fish.

Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization.

During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed.

Continuous nucleus extraction by optically-induced cell lysis on a batch-type microfluidic platform.

The extraction of a cell's nucleus is an essential technique required for a number of procedures, such as disease diagnosis, genetic replication, and animal cloning. However, existing nucleus extraction techniques are relatively inefficient and labor-intensive. Therefore, this study presents an innovative, microfluidics-based approach featuring optically-induced cell lysis (OICL) for nucleus extraction and collection in an automatic format. In comparison to previous micro-devices designed for nucleus extraction, the new OICL device designed herein is superior in terms of flexibility, selectivity, and efficiency. To facilitate this OICL module for continuous nucleus extraction, we further integrated an optically-induced dielectrophoresis (ODEP) module with the OICL device within the microfluidic chip. This on-chip integration circumvents the need for highly trained personnel and expensive, cumbersome equipment. Specifically, this microfluidic system automates four steps by 1) automatically focusing and transporting cells, 2) releasing the nuclei on the OICL module, 3) isolating the nuclei on the ODEP module, and 4) collecting the nuclei in the outlet chamber. The efficiency of cell membrane lysis and the ODEP nucleus separation was measured to be 78.04 ± 5.70% and 80.90 ± 5.98%, respectively, leading to an overall nucleus extraction efficiency of 58.21 ± 2.21%. These results demonstrate that this microfluidics-based system can successfully perform nucleus extraction, and the integrated platform is therefore promising in cell fusion technology with the goal of achieving genetic replication, or even animal cloning, in the near future.

Model Membrane and Cell Studies of Antimicrobial Activity of Melittin Analogues.

Melittin is a 26 residue peptide and the major component of bee (Apis mellifera) venom. Although melittin has both anticancer and antimicrobial properties, utilization has been limited due to its high lytic activity against eukaryotic cells. The mechanism of this lytic activity remains unclear but several mechanisms have been proposed, including pore formation or a detergent like mechanism, which result in lysis of cell membranes. Several analogues of melittin have been synthesized to further understand the role of specific residues in its antimicrobial and lytic activity. Melittin analogues that have a proline residue substituted for an alanine, lysine or cysteine have been studied with both model membrane systems and living cells. These studies have revealed that the proline residue plays a critical role in antimicrobial activity and cytotoxicity. Analogues lacking the proline residue and dimers of these analogues displayed decreased cytotoxicity and minimum inhibition concentrations. Several mutant studies have shown that, when key substitutions are made, the resultant peptides have more activity in terms of pore formation than the native melittin. Designing analogues that retain antimicrobial and anticancer activity while minimizing haemolytic activity will be a promising way to utilize melittin as a potential therapeutic agent.

Mechanistic investigation of interactions between steroidal saponin digitonin and cell membrane models.

Digitonin is an amphiphilic steroidal saponin, a class of natural products that can bind to cholesterol and lyse cells. Despite the known cell membrane lysis activity, it remains unclear how it interacts with cell membranes. In the present work, the interaction mechanism between digitonin and cell membrane models has quantitatively been investigated using a combination of physical techniques. It has been demonstrated that digitonin molecules bind specifically to cholesterol in the membrane, resulting in the formation of cholesterol-digitonin complexes on the membrane surface by removing cholesterol from the membrane core. Changes in the mass density and the film mechanics caused by the digitonin were determined by using quartz crystal microbalance with dissipation (QCM-D), and the combination of X-ray reflectivity (XRR) and dual polarization interferometry (DPI) yielded the hydration level of the cholesterol-digitonin complexes. From differential scanning calorimetry (DSC) analysis, supporting evidence was obtained that cholesterol was removed from the membrane core.

Curcumin reverse methicillin resistance in Staphylococcus aureus.

Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., was shown to possess superior potency to resensitize methicillin-resistant Staphylococcus aureus (MRSA) to antibiotics. Previous studies have shown the synergistic activity of curcumin with β-lactam and quinolone antibiotics. Further, to understand the anti-MRSA mechanism of curcumin, we investigated the potentiated effect of curcumin by its interaction in diverse conditions. The mechanism of anti-MRSA action of curcumin was analyzed by the viability assay in the presence of detergents, ATPase inhibitors and peptidoglycan (PGN) from S. aureus, and the PBP2a protein level was analyzed by western blotting. The morphological changes in the curcumin-treated MRSA strains were investigated by transmission electron microscopy (TEM). We analyzed increased susceptibility to MRSA isolates in the presence of curcumin. The optical densities at 600 nm (OD600) of the suspensions treated with the combinations of curcumin with triton X-100 and Tris were reduced to 63% and 59%, respectively, compared to curcumin without treatment. N,N'-dicyclohexylcarbodiimide (DCCD) and sodium azide (NaN3) were reduced to 94% and 55%, respectively. When peptidoglycan (PGN) from S. aureus was combined with curcumin, PGN (0–125 μg/mL) gradually blocked the antibacterial activity of curcumin (125 μg/mL); however, at a concentration of 125 µg/mL PGN, it did not completely block curcumin. Curcumin has a significant effect on the protein level of PBP2a. The TEM images of MRSA showed damage of the cell wall, disruption of the cytoplasmic contents, broken cell membrane and cell lysis after the treatment of curcumin. These data indicate a remarkable antibacterial effect of curcumin, with membrane permeability enhancers and ATPase inhibitors, and curcumin did not directly bind to PGN on the cell wall. Further, the antimicrobial action of curcumin involved in the PBP2a-mediated resistance mechanism was investigated.

NaD1 forms oligomeric complexes with phosphatidylinositol to lyse cell membranes

Cationic antimicrobial peptides (CAPs) are innate defense molecules produced by essentially all living species and represent a rich source of novel therapeutic molecules for the treatment of human diseases including cancer. Although several CAPs (mainly from amphibians and vertebrates) have been shown to exhibit direct toxicity against tumor cells, the underlying molecular mechanisms are not well understood. Nicotiana alata (ornamental tobacco) defensin 1, NaD1, is a Class II solanaceous plant defensin in the CAP family that exhibits antifungal activities. We have identified NaD1 as a potent molecule in killing mammalian tumour cells. Microscopy analyses have revealed that NaD1 induces cytolysis of tumor cells via the formation of large plasma membrane blebs. NaD1 was demonstrated to bind directly to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 or PIP2) resulting in disruption of cytoskeleton-membrane interactions and subsequent plasma membrane blebbing. To define the molecular basis of the interaction between NaD1 and PIP2, we determined the crystal structure of a NaD1:PIP2 complex. Strikingly, NaD1 forms a unique arch-shaped oligomer comprised of seven NaD1 dimers and 14 PIP2. The structure of the protein:lipid complex indicates that the presence of PIP2 is critical for oligomerisation of NaD1. Formation of NaD1:PIP2 oligomers was further confirmed by protein-protein crosslinking and transmission electron microscopy. Based on these data we propose that NaD1 is an innate pattern recognition molecule for PIP2 and forms a unique protein-lipid oligomeric complex that mediates permeabilisation of both microbes and tumour cells.

Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane

Gram-negative Escherichia coli (ATCC 8735) and Gram-positive Staphylococcus aureus (ATCC 3101) were selected as model bacteria to determine the antimicrobial mechanism of cinnamaldehyde. Several techniques were utilized to investigate the effects of cinnamaldehyde on food-borne bacterial membranes. The ultraviolet (UV) absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity. β-Galactosidase activity was determined to detect inner membrane permeability. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to observe bacterial morphology. Samples from both strains exposed to cinnamaldehyde showed higher UV absorptions, conductivity values, and β-Galactosidase activities compared with the control group and displayed a rapid rise trend. Thereafter, the values stabilized at a relatively steady state. SEM shows that treated E. coli and S. aureus cell samples exhibited rough cell membranes with particulate matter, and some of the S. aureus cells split due to deep wrinkle formation and distortion, unlike the control group. TEM shows that the bacteria treated with cinnamaldehyde exhibited numerous abnormalities, including cytoplasmic membrane separation from the cell wall, cell wall and cell membrane lysis, cytoplasmic content leakage, cytoplasmic content polarization, cell distortion, and cytoplasmic content condensation. These results indicate that bacterial cell morphology, membrane integrity, and permeability are damaged when the E. coli and S. aureus cells are exposed to the minimum inhibitory concentrations of cinnamaldehyde (0.31 mg/mL). In addition, the higher the cinnamaldehyde concentration, the more serious the bacterial membrane damage is.

An optimized protocol for isolating primary epithelial cell chromatin for ChIP.

A critical part of generating robust chromatin immunoprecipitation (ChIP) data is the optimization of chromatin purification and size selection. This is particularly important when ChIP is combined with next-generation sequencing (ChIP-seq) to identify targets of DNA-binding proteins, genome-wide. Current protocols refined by the ENCODE consortium generally use a two-step cell lysis procedure that is applicable to a wide variety of cell types. However, the isolation and size selection of chromatin from primary human epithelial cells may often be particularly challenging. These cells tend to form sheets of formaldehyde cross-linked material in which cells are resistant to membrane lysis, nuclei are not released and subsequent sonication produces extensive high molecular weight contamination. Here we describe an optimized protocol to prepare high quality ChIP-grade chromatin from primary human bronchial epithelial cells. The ENCODE protocol was used as a starting point to which we added the following key steps to separate the sheets of formaldehyde-fixed cells prior to lysis. (1) Incubation of the formaldehyde-fixed adherent cells in Trypsin-EDTA (0.25% room temperature) for no longer than 5 min. (2) Equilibration of the fixed cells in detergent-free lysis buffers prior to each lysis step. (3) The addition of 0.5% Triton X-100 to the complete cell membrane lysis buffer. (4) Passing the cell suspension (in complete cell membrane lysis buffer) through a 25-gauge needle followed by continuous agitation on ice for 35 min. Each step of the modified protocol was documented by light microscopy using the Methyl Green-Pyronin dual dye, which stains cytoplasm red (Pyronin) and the nuclei grey-blue (Methyl green). This modified method is reproducibly effective at producing high quality sheared chromatin for ChIP and is equally applicable to other epithelial cell types.