Research progress in infection-associated virulence factors of Staphylococcus aureus / 中华临床感染病杂志

Staphylococcus aureus is an important pathogen of human infectious diseases, which can cause skin and soft tissue infections, endocarditis, necrotizing pneumonia, myelitis and other serious infectious diseases. With the use of antibiotics, Staphylococcus aureus is evolving to develop drug resistance; at the same time it produces a variety of virulence factors to attack the host. This article will review the recent advances of Staphylococcus aureus virulence factors associated with the three stages of infection and introduce the detection methods of virulence factors briefly.

Development and optimization of cosolvent-based blended Sertraline orodispersible films - A step to personalized medicine

Abstract Personalized medicine is gaining importance in pharmacotherapeutics as it allows tailoring the drug treatment to achieve the best patient response. Orodispersible film (ODF) is easy to formulate in hospitals, produces dose flexibility to suit an individual needs, particularly for patients suffer from swallowing issues or prohibited to take fluids. Sertraline Hydrochloride (SRT) was solubilized in several cosolvents, then different SRT ODFs based on five hydrophilic polymers namely; polyvinyl alcohol (PVA), hydroxylethyl cellulose (HEC), hydroxypropyl methylcellulose E5 LV (HPMC E5 LV), sodium alginate (NaAlg) and gelatin at two concentrations (2% and 4%) were developed and characterized. The outcomes were exposed to response surface analysis to obtain the desirability results to obtain the optimized formulation. Blended ODFs were developed from 4% PVA and 2% HEC in different blends and then potassium chloride (KCl) as a pore-forming agent was added to the best formulation to investigate its dissolution enhancement effect. F14 containing 4% PVA: 2% HEC 2:1 with 5% KCl showed best physicochemical properties of suitable pH (5.6), disintegration time (6 sec), good folding endurance which released 91 % SRT after 15 min. SRT ODF is an encouraging delivery system in the course of personalized medicine for the management of depression.

Improvement on the tableting properties of traditional Chinese medicine extracts by fluid-bed coating and pore forming / 药学学报

This paper aimed to study the effect of combined co-processing of coating and pore forming on the tableting and tablet properties of traditional Chinese medicine (TCM) extracts together with its applicability. Four TCM extracts were co-processed using fluid bed with hydroxypropyl methyl cellulose (HPMC) as coating agent and ammonium bicarbonate (NH4HCO3) as pore-forming agent. Powder properties (such as particle size and size distribution, bulk density, tap density, moisture content) and tablet properties (including tensile strength, compaction ratio, fast elastic stretch, and disintegration time) were measured and compared among the powders. Scanning electron microscopy (SEM) was applied to characterize the surface of particles and tablets. Results showed that the particle size, flowability, and compactibility of the composite particles with HPMC were superior to the parent powders of TCM extracts. These properties of the porous particles with HPMC and NH4HCO3 showed further improvements. In addition, the addition of HPMC prolonged the disintegration time of tablets, whereas the pore-forming effect of NH4HCO3 could shorten the disintegration time. SEM revealed the changes in the morphology of the composite particles and the pores on the surface of the porous particles and tablets. In conclusion, co-processing with HPMC and NH4HCO3 could improve the powder and tablet properties of TCM extract powders, and this method shows certain applicability, which provides a feasible choice for improving the tableting properties of some TCM extract powders.

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.(AU)

Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin

Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.

Research progresses of the effective mechanism of pyroptosis and its related diseases / 医学研究生学报

Pyroptosis is a newly discovered form of programmed cell death, which has a close relationship with infectious diseases and spontaneous inflammatory disease and autoimmune disease.Gasdermin-D is the effector of pyroptosis.Inflammatory caspases cleave Gasdermin-D, break the structural autoinhibition and active the pore-forming activity of Gasdermin-D.Gasdermin-D-N domain is the active form of Gasdermin-D.Oligomerized Gasdermin-D-N domain forms membrane pores.Both pyroptosis and necroptosis can form pores.In this article, the progress of the research on pyroptosis, the involvement of Gasdermin-D in the molecular mechanism of pyroptosis, the distinguishment of pyroptosis from necroptosis and pyroptosis-related diseases are reviewed.

Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

The hemolytic activity of skin secretions obtained by stimulating the frog Kaloula pulchra hainana with diethyl ether was tested using human, cattle, rabbit, and chicken erythrocytes. The skin secretions had a significant concentration-dependent hemolytic effect on erythrocytes. The hemolytic activity of the skin secretions was studied in the presence of osmotic protectants (polyethylene glycols and carbohydrates), cations (Mg2+, Ca2+, Ba2+, Cu2+, and K+), or antioxidants (ascorbic acid, reduced glutathione, and cysteine). Results Depending on their molecular mass, osmotic protectants effectively inhibited hemolysis. The inhibition of skin hemolysis was observed after treatment with polyethylene glycols (1000, 3400, and 6000 Da). Among divalent cations, only 1 mM Cu2+ markedly inhibited hemolytic activity. Antioxidant compounds slightly reduced the hemolytic activity. Conclusions The results suggested that skin secretions of K. pulchra hainana induce a pore-forming mechanism to form pores with a diameter of 1.36-2.0 nm rather than causing oxidative damage to the erythrocyte membrane.

Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

Background: Previous works had shown that scorpion venom induced neurotransmitter elevation and an inflammatory response associated with various anatomo-pathological modifications. The most dangerous scorpions species in Algeria responsible for these effects are Androctonus australis hector (Aah) and Androctonus amoreuxi (Aam). Results: Comparison of the physiopathological effects induced by the two venoms showed differences in the kinetic of cytokine release and in lung injury. The lung edema was only observed in response to Aah venom and it was correlated with cell infiltration. In order to better understand the involved mechanism in inflammatory response, we used two antagonists, atropine (non-selective muscarinic antagonist) and propranolol (ß adrenergic antagonist), which lead to a decrease of cell infiltration but has no effect on edema forming. Conclusion: These results suggest another pathway in the development of lung injury following envenomation with Aam or Aah venom.(AU)

The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition.

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1–31 of St I (StI1-31) or 1–30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1–10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides’ abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.(AU)

A novel pore-forming hemolysin protein in Leptospira species / 中国人兽共患病学报

Hemolysins of Leptospira interragans have been shown to be the virulence factor in the pathogenesis of leptospirosis and 10 potential hemolysin genes were charecterized by genomic annotation of L.interrogans serovar.Lai strain 56601. In the present study, the LA0202 gene supposed to encode one of the new potential hemolysin was cloned and the protein encoded was purified. The purified protein was shown to have highly hemolytic activity as demonstrated on the sheep blood agar plate. It was also confirmed that the LA0202 protein-mediated hemolysis on sheep erythrocytes was osmotically protected by PEG6000. Meanwhile, this protein could induce pore formation on sheep erythrocytes and cause damages on the membrane of human L-02 liver cells. In addition, it could induce apoptosis of human L-02 liver cells after treatment of cells with this protein for 24 hours. It is evident that LA0202 protein acting as a pore-formong hemolysin can induce cytotoxic damage on mammalian cells.