Collagen adhesin–nanoparticle interaction impairs adhesin's ligand binding mechanism

Background

Pathogenic bacteria specifically recognize extracellular matrix (ECM) molecules of the host (e.g. collagen, fibrinogen and fibronectin) through their surface proteins known as MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) and initiate colonization. On implantation, biomaterials easily get coated with these ECM molecules and the MSCRAMMs mediate bacterial adherence to biomaterials. With the rapid rise in antibiotic resistance, designing alternative strategies to reduce/eliminate bacterial colonization is absolutely essential.

Methods

The Rhusiopathiae surface protein B (RspB) is a collagen‐binding MSCRAMM of Erysipelothrix rhusiopathiae. It also binds to abiotic surfaces. The crystal structure of the collagen‐binding region of RspB (rRspB_31–348) reported here revealed that RspB also binds collagen by a unique ligand binding mechanism called “Collagen Hug” which is a common theme for collagen‐binding MSCRAMMs of many Gram-positive bacteria. Here, we report the interaction studies between rRspB_31–348 and silver nanoparticles using methods like gel shift assay, gel permeation chromatography and circular dichroism spectroscopy.

Results

The “Collagen Hug” mechanism was inhibited in the presence of silver nanoparticles as rRspB_31–348 was unable to bind to collagen. The total loss of binding was likely because of rRspB_31–348 and silver nanoparticle protein corona formation and not due to the loss of the structural integrity of rRspB_31–348 on binding with nanoparticles as observed from circular dichroism experiments.

General significance

Interaction of rRspB_31–348 with silver nanoparticle impaired its ligand binding mechanism. Details of this inhibition mechanism may be useful for the development of antimicrobial materials and antiadhesion drugs.     

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.     

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.     

Design,synthesis and antimicrobial evaluation of novel 1-benzyl 2-butyl-4-chloroimidazole embodied 4-azafluorenones via molecular hybridization approach

A series of novel 1-benzyl-2-butyl-4-chloroimidazole embodied 4-azafluorenone hybrids, designed via molecular hybridization approach, were synthesized in very good yields using one pot condensation of 1-benzyl-2-butyl-4-chloroimidazole-5-carboxaldehyde, 1,3-indanedione, aryl/heteroaryl methyl ketones and ammonium acetate. All the synthetic derivatives were fully characterized by spectral data and evaluated for antimicrobial activity by disc diffusion method against selected bacteria and fungal strains. Among the 15 new compounds screened, 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(furan-2-yl)-5H-indeno[1,2-b]pyridin-5-one(10k) has pronounced activity with higher zone of inhibition (ZoI) against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Aspergillus flavus and Candida albicans. Also 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(dibenzo[b,d]thiophen-2-yl)-5H-indeno [1,2-b]pyridin-5-one (10n) and 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(3-tosyl-3H-inden-1-yl)-5H-indeno[1,2-b]pyridin-5-one (10o) showed selective higher inhibitory activity against Aspergillus flavus and Candida albicans. The results demonstrated potential importance of molecular hybridization in the development of 10k as potential antimicrobial agent.     

Dynamic changes to the endocannabinoid system in models of chronic pain

The analgesic effects of cannabinoid ligands, mediated by CB1 receptors are well established. However, the side-effect profile of CB1 receptor ligands has necessitated the search for alternative cannabinoid-based approaches to analgesia. Herein, we review the current literature describing the impact of chronic pain states on the key components of the endocannabinoid receptor system, in terms of regionally restricted changes in receptor expression and levels of key metabolic enzymes that influence the local levels of the endocannabinoids. The evidence that spinal CB2 receptors have a novel role in the modulation of nociceptive processing in models of neuropathic pain, as well as in models of cancer pain and arthritis is discussed. Recent advances in our understanding of the spinal location of the key enzymes that regulate the levels of the endocannabinoid 2-AG are discussed alongside the outcomes of recent studies of the effects of inhibiting the catabolism of 2-AG in models of pain. The complexities of the enzymes capable of metabolizing both anandamide (AEA) and 2-AG have become increasingly apparent. More recently, it has come to light that some of the metabolites of AEA and 2-AG generated by cyclooxygenase-2, lipoxygenases and cytochrome P450 are biologically active and can either exacerbate or inhibit nociceptive signalling.     

RNA interference mediated inhibition of dengue virus multiplication and entry in HepG2 cells

Background

Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells.

Methodology/Principal Findings

HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells.

Conclusions/Significance

Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS. Furthermore, a decrease of viremia in humans can result in the reduction of infected vectors and thus, halt of the transmission cycle.