Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.     

Extraction, amplification and characterization of wood DNA from dipterocarpaceae

A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood if suitable information on genetic variation patterns within and among species is available. Potential applications are in forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites.     

Correlation of C4ST-1 and ChGn-2 expression with chondroitin sulfate chain elongation in atherosclerosis

Subendothelial retention of lipoproteins by proteoglycans (PGs) is the initiating event in atherosclerosis. The elongation of chondroitin sulfate (CS) chains is associated with increased low-density lipoprotein (LDL) binding and progression of atherosclerosis. Recently, it has been shown that 2 Golgi enzymes, chondroitin 4-O-sulfotransferase-1 (C4ST-1) and chondroitin N-acetylgalactosaminyltransferase-2 (ChGn-2), play a critical role in CS chain elongation. However, the roles of C4ST-1 and ChGn-2 during the progression of atherosclerosis are not known. The aim of this study was to analyze the expression of C4ST-1 and ChGn-2 in atherosclerotic lesions in vivo and determine whether their expression correlated with CS chain elongation.Low-density lipoprotein receptor knockout (LDLr KO) mice were fed a western diet for 2, 4, and 8 weeks to stimulate development of atherosclerosis. The binding of LDL and CS PG in this mouse model was confirmed by chondroitinase ABC (ChABC) digestion and apolipoprotein B (apo B) staining. Gel filtration analysis revealed that the CS chains began to elongate as early as 2 weeks after beginning a western diet and continued as the atherosclerosis progressed. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the mRNA levels of C4ST-1 and ChGn-2 increased after 8 weeks of this diet. In contrast, the mRNA levels of their homologs, C4ST-2 and ChGn-1, were unchanged. In addition, immunohistochemical analysis demonstrated that the expression of C4ST-1 and ChGn-2 appeared to have similar site-specific patterns and coincided with biglycan expression at the aortic root.Our results suggested that C4ST-1 and ChGn-2 may be involved in the elongation of CS chains in the arterial wall during the progression of atherosclerosis. Therefore, modulating their expression and activity might be a novel therapeutic strategy for atherosclerosis.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins

Heterotrimeric G_i proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G_i proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of G_i2 or G_i1/3 protein in G_i2-knockout (G_i2–/–) or G_i1/3-knockout (G_i1/3–/–) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (M) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF- and IFN- in splenocytes from G_i2–/– mice compared with wild-type (WT) mice. Also, LPS-induced TNF- was increased in splenocytes from G_i1/3–/– mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-, IL-10, and thromboxane B₂ (TxB₂) production was decreased in M harvested from G_i2–/– mice. Also, LPS-induced production of IL-10 and TxB₂ was decreased in M from G_i1/3–/– mice. In subsequent in vivo studies, TNF- levels after LPS challenge were significantly greater in G_i2–/– mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated G_i2–/– mice compared with WT mice. These data suggest that G_i proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli. G_i protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors     

A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki

Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys⁸ to Cys¹⁶) was identified.     

E. coli sabotages the in vivo production of O-linked β-N-acetylglucosamine-modified proteins

The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.