Binding Capacity Of Membranes Research Articles

Human cytochrome C natural variants: Studying the membrane binding properties of G41S and Y48H by fluorescence energy transfer and molecular dynamics

Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally occurring mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4. In this study, we have investigated the binding of such variants and of wild type (wt) cyt C to synthetic cardiolipin-containing vesicles. The mutants have a lower propensity in membrane binding, displaying higher dissociation constants with respect to the wt protein. Compressibility measurements reveal that both variants are more flexible than the wt, suggesting that the native central Ω-loop is important for the interaction with membranes. Such hypothesis is supported by molecular dynamics simulations. A minimal distance analysis indicates that in the presence of cardiolipin the central Ω-loop of the mutants is no more in contact with the membrane, as it happens instead in the case of wt cyt C. Such finding might provide a hint for the reduced membrane binding capacity of the variants and their enhanced peroxidase activity in vivo.

Sphingosine-1-phosphate Decreases Erythrocyte Dysfunction Induced by β-Amyloid.

Amyloid beta peptides (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Soluble Aβ oligomers, rather than monomer or insoluble amyloid fibrils, show red blood cell (RBC) membrane-binding capacity and trigger several morphological and functional alterations in RBCs that can result in impaired oxygen transport and delivery. Since bioactive lipids have been recently proposed as potent protective agents against Aβ toxicity, we investigated the role of sphingosine-1-phosphate (S1P) in signaling pathways involved in the mechanism underlying ATP release in Ab-treated RBCs. In RBCs following different treatments, the ATP, 2,3 DPG and cAMP levels and caspase 3 activity were determined by spectrophotometric and immunoassay. S1P rescued the inhibition of ATP release from RBCs triggered by Ab, through a mechanism involving caspase-3 and restoring 2,3 DPG and cAMP levels within the cell. These findings reveal the molecular basis of S1P protection against Aβ in RBCs and suggest new therapeutic avenues in AD.

Computational design and genetic incorporation of lipidation mimics in living cells.

Protein lipidation, which regulates numerous biological pathways and plays crucial roles in the pharmaceutical industry, is not encoded by the genetic code but synthesized post-translationally. In the present study, we report a computational approach for designing lipidation mimics that fully recapitulate the biochemical properties of natural lipidation in membrane association and albumin binding. Furthermore, we establish an engineered system for co-translational incorporation of these lipidation mimics into virtually any desired position of proteins in Escherichia coli and mammalian cells. We demonstrate the utility of these length-tunable lipidation mimics in diverse applications, including improving the half-life and activity of therapeutic proteins in living mice, anchoring functional proteins to membrane by substituting natural lipidation, functionally characterizing proteins carrying different lengths of lipidation and determining the plasma membrane-binding capacity of a given compound. Our strategy enables gain-of-function studies of lipidation in hundreds of proteins and facilitates the creation of superior therapeutic candidates.

A facile and selective derivatization approach on kynurenine-NH2 in daptomycin, leading to the discovery of hexakynomycin to combat multidrug-resistant Gram-positive pathogens especially daptomycin-resistant bacteria

Wide-spread use of daptomycin unavoidably resulted in the emergence of daptomycin-resistant pathogens. In the hunt for more active daptomycin derivatives through medicinal chemistry studies, we established a concise semisynthetic approach to modify the L-Kyn13 on daptomycin specifically and effectively. Here, 19 novel derivatives with certain diversity were designed and synthesized to perform a comprehensive SAR study on this underestimated position. The optimal compound, termed “hexakynomycin”, as the new generation of daptomycin-based antibiotic candidate exhibited 4->125-fold higher activity against methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-resistant Enterococci (VRE), including daptomycin-resistant strains, compared with that of daptomycin. Greater membrane binding capacity rendered hexakynomycin better activity and special antibiotic property. Hexakynomycin also demonstrated a better pharmacokinetic profile, good safety features and good pharmacodynamics properties. This work provided an effective modification strategy aiming at daptomycin which provided significant insights and showed great promise for the next generation of daptomycin derivatives.

Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane

A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate.

Regions of hepatitis C virus E2 required for membrane association

Hepatitis C virus (HCV) uses a hybrid entry mechanism. Current structural data suggest that upon exposure to low pH and Cluster of Differentiation 81 (CD81), the amino terminus of envelope glycoprotein E2 becomes ordered and releases an internal loop with two invariant aromatic residues into the host membrane. Here, we present the structure of an amino-terminally truncated E2 with the membrane binding loop in a bent conformation and the aromatic side chains sequestered. Comparison with three previously reported E2 structures with the same Fab indicates that this internal loop is flexible, and that local context influences the exposure of hydrophobic residues. Biochemical assays show that the amino-terminally truncated E2 lacks the baseline membrane-binding capacity of the E2 ectodomain. Thus, the amino terminal region is a critical determinant for both CD81 and membrane interaction. These results provide new insights into the HCV entry mechanism.

Separation and Identification of Native Proteoglycans by Composite Agarose-Polyacrylamide Gel Electrophoresis and Immunoblotting.

Composite agarose-polyacrylamide gel electrophoresis (CAPAGE) in gels of 1.2% w/v polyacrylamide and 0.6% w/v agarose can be used to examine the heterogeneity of full-length native proteoglycan populations and their fragments in crude tissue extracts, and when used in conjunction with immunoblotting and specific antibodies to proteoglycan core protein and glycosaminoglycan, side chain epitopes can provide significant information on the level of proteoglycan polydispersity/heterogeneity and a number of proteoglycan populations present in tissue samples. This can be a technically difficult technique, but it reveals significant information on proteoglycans from small tissue samples not possible by any other separation methodology. Native full-length and proteoglycan fragments are examined in this technique something which cannot be done in the popular SDS-PAGE format unless the glycosaminoglycan side chains are first removed. Furthermore, since proteoglycans do not require renaturation from SDS-protein complexes, the proteoglycan populations separated by native electrophoresis are highly reactive with antibodies in immunoblotting procedures. Despite the massive sizes of proteoglycans, transfer conditions have been determined which provide close to quantitative transfer to nitrocellulose membranes without exceeding the binding capacity of such membranes, avoiding bleed-through of the transferred proteoglycans. Development of biotinylated hyaluronan and its application in an affinity blotting procedure has also yielded significant information on aggregatable proteoglycan populations separated by CAPAGE from a number of cartilages and vascular tissues in health and disease. While the CAPAGE system can be a technically demanding technique to master particularly in gel preparation, all other steps are straightforward, and the method yields invaluable information on proteoglycan populations extracted from connective tissues in health and disease that cannot be ascertained by any other technique. Further improvements in the detection of proteoglycan features with the development of novel bio-affinity probes or new antibody preparations are expected to further improve the utility of CAPAGE separation methodology.

Cys mutants as tools to study the oligomerization of the pore-forming toxin sticholysin I

Sticholysin I (StI) is a water-soluble protein with the ability to bind membranes where it oligomerizes and forms pores leading to cell death. Understanding the assembly property of this protein may be valuable for designing potential biotechnological tools, such as stable or structurally defined nanopores. In order to get insights into the stabilization of StI oligomers by disulfide bonds, we designed and characterized single and double cysteine mutants at the oligomerization interface. The oligomer formation was induced in the presence of lipid membranes and visualized by SDS-PAGE. The contribution of the oligomeric structures to the membrane binding and pore-forming capacities of StI was assessed. Single and double cysteine introduction at the protein-protein oligomerization interface does not considerably affect the conformation and function of the monomeric protein. In the presence of membranes, a cysteine double mutation at positions 15 and 59 favored formation of different size oligomers stabilized by disulfide bonds. The results of this work highlight the relevance of these positions (15 and 59) to be considered for developing biosensors based on nanopores from StI.

Phosphate-Based Reactive Membranes for Uranium Isotopic Screening

This study describes the application of reactive membranes for the isolation and concentration of uranium from circumneutral pH solutions by ultrafiltration. The reactive membranes were prepared by grafting a uranium-selective polymer layer via ultraviolet-initiated radical polymerization from the surfaces of poly(ethersulfone) ultrafiltration membrane supports. Dynamic uranium binding capacity measurements were conducted using the reactive membranes housed in an inline filter column, and column breakthrough data were fitted to theoretical models. The experimental data were best described by the Thomas model, indicating that uranium sorption was a reaction rate-limited process. Fitted model parameter values were compared with the results from batch experiments, where similar reaction rate constants were obtained for loading from solutions at pH 4 and 6. The maximum uranium binding capacity of the reactive membrane decreased in the presence of multiple competing ions, from 7.8 ± 0.3 mg g–1 in deionized water to 3.6 ± 0.2 mg g–1 in simulated seawater at pH 6. Alpha spectroscopy pulse-height spectra of uranium-loaded reactive membranes were analyzed. Peak energy resolutions measured as full width at half-maximum of 70 ± 8 and 65 ± 5 keV were obtained from samples loaded with uranium from DI and seawater solutions. The results of this study provide an approach for a rapid, on-field screening of liquid samples to complement existing techniques and accelerate sample analyses.

Sphingosine 1-phosphate attenuates neuronal dysfunction induced by amyloid-β oligomers through endocytic internalization of NMDA receptors.

Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aβ (Aβ42 O) show membrane-binding capacity and trigger the disruption of Ca2+ homeostasis, a causative event in neuron degeneration. Since bioactive lipids have been recently proposed as potent protective agents against Aβ toxicity, we investigated the involvement of sphingosine 1-phosphate (S1P) signalling pathway in Ca2+ homeostasis in living neurons exposed to Aβ42 O. We show that both exogenous and endogenous S1P rescued neuronal Ca2+ dyshomeostasis induced by toxic Aβ42 O in primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Further analysis revealed a strong neuroprotective effect of S1P1 and S1P4 receptors, and to a lower extent of S1P3 and S1P5 receptors, which activate the Gi -dependent signalling pathways, thus resulting in the endocytic internalization of the extrasynaptic GluN2B-containing N-methyl-D-aspartate receptors (NMDARs). Notably, the S1P beneficial effect can be sustained over time by sphingosine kinase-1 overexpression, thus counteracting the down-regulation of the S1P signalling induced by Aβ42 O. Our findings disclose underlying mechanisms of S1P neuronal protection against harmful Aβ42 O, suggesting that S1P and its signalling axis can be considered promising targets for therapeutic approaches for AD.

ANXA2 Facilitates Enterovirus 71 Infection by Interacting with 3D Polymerase and PI4KB to Assist the Assembly of Replication Organelles.

Similar to that of other enteroviruses, the replication of enterovirus 71 (EV71) occurs on rearranged membranous structures called replication organelles (ROs). Phosphatidylinositol 4-kinase III (PI4KB), which is required by enteroviruses for RO formation, yields phosphatidylinositol-4-phosphate (PI4P) on ROs. PI4P then binds and induces conformational changes in the RNA-dependent RNA polymerase (RdRp) to modulate RdRp activity. Here, we targeted 3D polymerase, the core enzyme of EV71 ROs, and found that the host factor Annexin A2 (ANXA2) can interact with 3D polymerase and promote the replication of EV71. Then, an experiment showed that the annexin domain of ANXA2, which possesses membrane-binding capacity, mediates the interaction of ANXA2 with EV71 3D polymerase. Further research showed that ANXA2 is localized on ROs and interacts with PI4KB. Overexpression of ANXA2 stimulated the formation of PI4P, and the level of PI4P was decreased in ANXA2-knockout cells. Furthermore, ANXA2, PI4KB, and 3D were shown to be localized to the viral RNA replication site, where they form a higher-order protein complex, and the presence of ANXA2 promoted the PI4KB-3D interaction. Altogether, our data provide new insight into the role of ANXA2 in facilitating formation of the EV71 RNA replication complex.

Spongy membranes for peroxidase purification from Brassica oleracea roots

In this work, it was aimed to purify peroxidase from Brassica oleracea roots using newly synthesized cryogel membranes. 2-4-vinylphenylboronic acid (VPBA) containing 2-hydroxyethyl methacrylate (HEMA) [PHEMABA] cryogel membranes were prepared successfully and swelling tests, scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy, contact angle measurements, and Brauner-Emmet-Teller (BET) surface area analysis were done for characterization. Effect of experimental conditions on peroxidase binding capacity of PHEMABA cryogel membranes was determined by examining parameters such as pH, temperature and time with binding studies. Maximum peroxidase binding capacity of PHEMABA cryogel membranes was determined as 22.01 mg/g for 2 mg/mL equilibrium peroxidase concentration at pH 8.0. Finally, peroxidase was purified from Brassica oleracea roots with 85.38% yield and 18.38 purification fold. According to obtained results, spongy PHEMABA cryogel membranes show high peroxidase binding capacity and could be suggested as a boronate affinity model system for enzyme purification.

Highly efficient dye removal and lysozyme purification using strong and weak cation-exchange nanofiber membranes

Electrospinning technology was applied for the preparation of polyacrylonitrile (PAN) nanofiber membrane in this work. After hot pressing, alkaline hydrolysis and neutralization treatment, a weak acid cation exchange membrane (P-COOH) was prepared. By the covalent coupling reaction between the acidic membrane and aminomethane sulfonic acid (AMSA), a strong acidic nanofiber membrane (P-SO3H) was obtained. The surface morphology, chemical structure, and thermal stability of the prepared ion exchange membranes were analyzed via SEM, FTIR and TGA. Analytical results showed that the membranes were prepared successfully and thermally stable. The ion exchange membrane (IEX) was conducted with the newly designed membrane reactor, and different operating conditions affecting the adsorption efficiency of Toluidine Blue dye (TBO) were investigated by dynamic flow process. The results showed that dynamic binding capacity (DBC) of weak and strong IEX membranes for TBO dye was ~170 mg/g in a dynamic flow process. Simultaneously, the ion exchange membranes were also used for purifying lysozyme from chicken egg white (CEW). Results illustrated that the recovery yield and purification factor of lysozyme were 93.43% and 29.23 times (P-COOH); 90.72% and 36.22 times (P-SO3H), respectively. It was revealed that two type ion exchange membranes were very suitable as an adsorber for use in dye waste treatment and lysozyme purification process. P-SO3H strong ion-exchange membrane was more effective either removal of TBO dye or purification of lysozyme. The ion exchange membranes not only effectively purified lysozyme from CEW solution, but also effectively removed dye from wastewater.

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25mg would be tested, starting with 10mg in cohort 1. No significant difference between Mirococept at 10mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80mg (equivalent to 120mg in human kidney) that provides a basis for further clinical development.

Redundant and Diversified Roles Among Selected Arabidopsis thaliana EXO70 Paralogs During Biotic Stress Responses.

The heterooctameric vesicle-tethering complex exocyst is important for plant development, growth, and immunity. Multiple paralogs exist for most subunits of this complex; especially the membrane-interacting subunit EXO70 underwent extensive amplification in land plants, suggesting functional specialization. Despite this specialization, most Arabidopsis exo70 mutants are viable and free of developmental defects, probably as a consequence of redundancy among isoforms. Our in silico data-mining and modeling analysis, corroborated by transcriptomic experiments, pinpointed several EXO70 paralogs to be involved in plant biotic interactions. We therefore tested corresponding single and selected double mutant combinations (for paralogs EXO70A1, B1, B2, H1, E1, and F1) in their two biologically distinct responses to Pseudomonas syringae, root hair growth stimulation and general plant susceptibility. A shift in defense responses toward either increased or decreased sensitivity was found in several double mutants compared to wild type plants or corresponding single mutants, strongly indicating both additive and compensatory effects of exo70 mutations. In addition, our experiments confirm the lipid-binding capacity of selected EXO70s, however, without the clear relatedness to predicted C-terminal lipid-binding motifs. Our analysis uncovers that there is less of functional redundancy among isoforms than we could suppose from whole sequence phylogeny and that even paralogs with overlapping expression pattern and similar membrane-binding capacity appear to have exclusive roles in plant development and biotic interactions.

Radiation induced in-situ synthesis of membranes for removal of 2,4-dichlorophenoxy acetic acid from real water samples

A molecularly imprinted membrane for specific removal of 2,4-Dichlorophenoxyacetic acid (2,4-D) was synthesized by γ-initiated polymerization using N-vinyl imidazole as functional monomer and ethylene glycol dimethacrylate (EGDMA) as a crosslinker in methanol:water (1:4 by volume) mixture at room temperature. Functional monomer to template molecule ratio was kept as 4:1. The binding capacity of molecularly imprinted membranes was obtained as 75% within 100 min for 5 mg/L 2,4-D solution (on 25 mg of imprinted polymer). Binding characteristics in various template molecule concentrations and pH values were studied. Furthermore, the selectivity of the imprinted membranes was evaluated by using structurally similar herbicides such as (R)-2-(2,4-dichlorophenoxy) propanoic acid (2,4-DP) and 4-chlorophenoxyacetic acid (4-CPA). The binding capacity of membrane was investigated by using real water samples. In order to investigate the free volume size of the membranes positron annihilation lifetime spectroscopy (PALS) experiments were carried out.

Antimicrobial activity and mechanism of peptide CM4 against Pseudomonas aeruginosa.

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells and may possibly be used as an antimicrobial agent. In this study, a C-terminal amidated antibacterial peptide ABP-CM4 (ABP-CM4N) with the strongest antibacterial activity was obtained through screening the antibacterial activities of ABP-CM4 with different modifications. The minimal inhibitory concentration of ABP-CM4N was 8 μM against P. aeruginosa (ATCC 27853) which was lower than that of ABP-CM4 (16 μM). The strengthened antimicrobial activity of ABP-CM4N may be associated with the increased membrane binding capacity, being two times that of ABP-CM4 (p < 0.001). The antibacterial mechanism of ABP-CM4N to Pseudomonas aeruginosa was examined by means of cell membrane integrity analysiss, the intracellular ultrastructure change observation and E. coli genomic DNA binding assay. It was found that ABP-CM4N had the same antimicrobial mechanism as ABP-CM4, and the aim of the antimicrobial mechanism was mainly to destroy the cell membrane which caused nucleic acid or protein leakage, and secondly to interact with E. coli genomic DNA after penetrating the cell membrane. Furthermore, in vitro ABP-CM4N showed a better bacteriostatic activity in meats, with the treated samples showing two to three times less positive colonies than ABP-CM4.

Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

ABSTRACT Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of T. pyogenes. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1β, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO. Abbreviations: T. pyogenes: Trueperella pyogenes; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-β-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin–Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1β: interleukin-1β; IFN-γ: interferon-γ; TGF-β: transforming growth factor-β; ELISA: enzyme-linked immunosorbent assay

Correction to: α-Synuclein's Uniquely Long Amphipathic Helix Enhances its Membrane Binding and Remodeling Capacity.

The original version of the article unfortunately contained error in author group; two authors were not submitted and published in the original version. Also the funding information is erroneously omitted.

Improved expression of green fluorescent protein in cattle embryos produced by ICSI-mediated gene transfer with spermatozoa treated with streptolysin-O

The ICSI-sperm mediated gene transfer (ICSI-SMGT) has been used to produce transgenic mice with high efficiency; however, the efficiency of this technique in farm animals is still less than desirable. Pretreatment of sperm with membrane destabilizing agents can improve the efficiency of ICSI in cattle. The objective of the present study was to evaluate streptolysin-O (SLO) as a novel treatment to permeabilize the bovine sperm membrane and assess its effect on efficiency of generating transgenic embryos by ICSI-SMGT. First, there was evaluation of the plasma membrane integrity (SYBR/PI), acrosome membrane integrity (PNA/FITC), DNA damage (TUNEL) and binding capacity of exogenous DNA (Nick Translation) in bull sperm treated with SLO. Subsequently, there was assessment of embryonic development and the efficiency in generating transgenic embryos with enhanced expression of the gene for green fluorescent protein (EGFP). Results indicate that SLO efficiently permeabilizes the plasma and acrosome membranes of bull spermatozoa and increases binding of exogenous DNA mostly to the post-acrosomal region and tail without greatly affecting the integrity of the DNA. Furthermore, treatment of bull spermatozoa with SLO prior to the injection of oocytes by ICSI-SMGT significantly increased the rate of embryo expression of the EGFP gene. Future experiments are still needed to determine the effect of this treatment on the development and transgene expression in fetuses and animals produced by ICSI-SMGT.