scientificprotocols

Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

Introduction

To determine the specificity of the microbial proteases in triggering the respiratory proteins to produce bactericidal ROS, we compared the level of bacterial clearance of protease-producing and non-producing strains of typical Gram-negative and Gram-positive bacteria. For Gram-negative bacteria, we chose Pseudomonas aeruginosa strain PAO-Iglewski which produces PAE (the major extracellular protease virulence factor), and the PAE-knockout mutant PAO-B1A1 which does not produce PAE (1). For Gram-positive bacteria, we cultured Staphylococcus aureus PC1839 strain which produces active V8-protease, and AK3 strain which is a V8 protease-mutant (2). The genes controlling PAE in P. aeruginosa and the V8-protease in S. aureus are under the control of quorum-sensing signals. Therefore the culture conditions which facilitate the production of active extracellular proteases are utilized.

Procedure

1. Inoculate single co

scientificprotocols

Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

Introduction

Lipopolysaccharide (LPS), also known as the pyrogen, resides on the outer membrane of the Gram-negative bacteria but not on the Gram-positive bacteria. Upon injection of the Gram-negative bacteria into the horseshoe crab, the LPS is necessary and sufficient to trigger the degranulation of the host hemocytes, which subsequently releases the intracellular factors to kill the invader effectively (1). Previous study demonstrated that the intracellular serine proteases, such as Factor B, pro-clotting enzyme, and clotting enzyme can activate the prophenoloxidase activity of the hemocyanin (HMC/PPO) to phenoloxidase (PO)2,3. To verify that the microbial extracellular protease could activate HMC/PPO to PO and further mediate the antimicrobial response in the extracellular milieu, Gram-positive bacteria which lack LPS and therefore do not trigger hemocyte degranulation are used in the in vivo antimicrobial assay. Being ubiquitous an

scientificprotocols

Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

Introduction

The interaction between HMC or metHb with LPS or LTA are examined by ELISA using LPS- or LTA-immobilized plates and probed with anti-hemocyanin or anti-hemoglobin antibody. To test the specificity of the ELISA, we further examined whether or not pre-incubation of HMC or Hb with LPS or LTA could reduce the ELISA-readout in a dose-dependent manner.

Procedure

1. Incubate aliquots of 100 μl LPS or LTA which is resuspended in 0.01 M pH 7.4 PBS in a 96-well microtitre plate (Maxisorp®, NUNC, Denmark) at room temperature overnight.

• Rinse the wells three times with 300 μl of PBS each.

scientificprotocols

Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

Introduction

To demonstrate the ability of microbial factor-activated PPO activity in clearing the invading pathogen in vivo, we infected horseshoe crabs in the presence or absence of PO-specific inhibitor, PTU (1) or kojic acid (2). A comparison of the remnant bacterial load under these conditions should help to clarify the specific contribution of PO, if any, to the antimicrobial activity. Previously, it was reported that HMC/PPO is activated by host intracellular factors released through LPS-dependent degranulation of hemocytes. To avoid provocation of PPO by such cellular components and to unequivocally demonstrate that the microbial factor-activated PPO contributes to the antimicrobial defense, Gram-positive bacteria lacking LPS were used to avoid LPS-induced hemocyte lysis. To this end, the S. aureus laboratory strains, PC1839 (V8 protease-producing) and AK3 (V8 protease inactive mutant), were injected into the animals.

Proc

scientificprotocols

Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

Introduction

The lysis of the red blood cells can be monitored by the release of hemoglobin to the extracellular environment. After removing the intact RBC by centrifugation, the absorbance at 404 nm of the supernatant reflects the hemolysis. The RBC lysate obtained by total disruption by 1% Triton- X100, is used as a positive control.

Procedure

The RBC lysis by bacterial hemolysin is measured as described1 with modification.

scientificprotocols

Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

Introduction

Azocoll, the Azo dye-impregnated collagen (Sigma) is used as a chromogenic non-specific substrate for protease activity assay. Upon proteolysis, soluble peptide fragments which is purple in color due to Azo dye-impregnation, are released and can be detected by absorbance at 550 nm.

Procedure

1. Resuspend 75 mg of Azocoll with 50 ml of 0.01 M PBS, pH 7.4.

• Gently swirl the suspension at room temperature for 2 h.

scientificprotocols

Authors: Naxin Jiang , Nguan Soon Tan , Bow Ho & Jeak Ling Ding 

Introduction

Hemoglobin is auto-oxidized from ferrous-hemoglobin to ferric hemoglobin (methemoglobin, metHb) with the production of H2O2. H2O2 initiates the catalytic cycle between the ferric (HbFeIII) and ferryl (HbFeIV) hemes, thus activating the pseudoperoxidase activity of metHb, which eliminates the H2O2, producing the superoxide (1). The chemiluminescence (CL) of Cypridina luciferin analog indicates the generation of O2•- or singlet oxygen, but not that of ozone, hydroxyl radicals or H2O22. To further confirm that the ROS species produced by metHb and indicated by CLA-CL is superoxide anions but not singlet oxygen, superoxide dismutase (SOD) is applied as a diagnostic confirmation of superoxide anion production

Procedure

The pseudoperoxidase activity is measured as described 2, with modifications.

scientificprotocols

Authors: Antoine Louveau & Jonathan Kipnis 

Abstract

Presented here is the method for dissection and immunostaining of the whole mount meninges from the mouse skullcap.

Introduction

Presented here is the method for dissection and immunostaining of the whole mount meninges from the mouse skullcap.

scientificprotocols

Authors: Laura Briñas, Céline Orvain, Caroline Belser, Corinne Cruaud, Karine Labadie, Laurie Bertrand, Valerie Barbe, Jean-Marc Aury, Patrick Wincker & Adriana Alberti 

Abstract

Bacterial artificial chromosome (BAC) libraries are still a valuable tool for de novo assembly of complex genomes, such as many plants genomes. Shotgun sequencing of BACs, individually or by pools, produces first assemblies which usually need further improvement towards finished quality. We developed a new approach to obtain BAC ends libraries for Illumina sequencing (BES), overcoming the expensive and time consuming BAC ends Sanger sequencing. This new method could be useful for improving de novo assembly, especially in the case of highly repeated genomes.

Introduction

Here, we describe an easily transposable method for BES library preparation and sequencing on Illumina platform from standard BACs libraries (Figure 1). DNA is mechanically fragmented and ends are repaired. After ligation of Cre-Lox adaptors, frag

scientificprotocols

Authors: Avantika Dhabaria, Paolo Cifani & Alex Kentsis 

Abstract

Miniaturization of columns for liquid chromatography has greatly enhanced its sensitivity and resolution. This protocol describes the fabrication of fused silica capillaries with frits and their packing with chromatography media for high-resolution, high-capacity separations suitable for mass spectrometry.

Reagents

Column fritting: