Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR

One of the major drawbacks of DNA-based microbial diagnostics is its inability to discriminate between live and dead bacteria. Due to the persistence of DNA in the environment after cells have lost their viability, DNA-based assays cannot assess pathogenic risk since signals can originate from both live and dead cells. Presented here is a potential application of the novel chemical propidium monoazide (PMA), which results in the selective suppression of DNA detection from dead cells. PMA can only penetrate dead cells with permeabilized cell membranes. Upon intercalation into the DNA, covalent crosslinkage of PMA to DNA is achieved through light exposure. This modification prevents the DNA from being amplified by PCR. The method, in combination with quantitative PCR as a diagnostic tool, successfully monitored the disinfection efficacy of hypochlorite, benzalkonium and heat on several model pathogens. Threshold cycle numbers increased with increasing disinfection strength after PMA treatment of samples compared to non-PMA treated samples. With some disinfectant-specific differences, monitoring viability loss with membrane integrity as an indicator seemed to be more conservative than monitoring viability loss with plate counts. Loss of viability after short UV-exposure could not be monitored with PMA as UV light affects viability by inducing DNA damage without directly affecting membrane permeability.     

Bioengineered human arginase I with enhanced activity and stability controls hepatocellular and pancreatic carcinoma xenografts

Hepatocellular carcinoma (HCC) and pancreatic carcinoma (PC) cells often have inherent urea cycle defects rendering them auxotrophic for the amino acid l-arginine (l-arg). Most HCC and PC require extracellular sources of l-arg and undergo cell cycle arrest and apoptosis when l-arg is restricted. Systemic, enzyme-mediated depletion of l-arg has been investigated in mouse models and human trials. Non-human enzymes elicit neutralizing antibodies, whereas human arginases display poor pharmacological properties in serum. Co(2+) substitution of the Mn(2+) metal cofactor in human arginase I (Co-hArgI) was shown to confer more than 10-fold higher catalytic activity (k(cat)/K(m)) and 5-fold greater stability. We hypothesized that the Co-hArgI enzyme would decrease tumor burden by systemic elimination of l-arg in a murine model. Co-hArgI was conjugated to 5-kDa PEG (Co-hArgI-PEG) to enhance circulation persistence. It was used as monotherapy for HCC and PC in vitro and in vivo murine xenografts. The mechanism of cell death was also investigated. Weekly treatment of 8 mg/kg Co-hArgI-PEG effectively controlled human HepG2 (HCC) and Panc-1 (PC) tumor xenografts (P = .001 and P = .03, respectively). Both cell lines underwent apoptosis in vitro with significant increased expression of activated caspase-3 (P < .001). Furthermore, there was evidence of autophagy in vitro and in vivo. We have demonstrated that Co-hArgI-PEG is effective at controlling two types of l-arg-dependent carcinomas. Being a nonessential amino acid, arginine deprivation therapy through Co-hArgI-PEG holds promise as a new therapy in the treatment of HCC and PC.     

A phospho-proteomic screen identifies novel S6K1 and mTORC1 substrates revealing additional complexity in the signaling network regulating cell growth

S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70^S6K1 isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85^S6K1 isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85^S6K1 substrates. Four novel putative p85^S6K1 substrates, GRP75, CCTβ, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTβ was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.     

Picomolar nitric oxide signals from central neurons recorded using ultrasensitive detector cells

Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time. Stimulation of NMDA receptors, which are coupled to activation of neuronal NO synthase, routinely generated NO signals from neurons in cerebellar slices. The average computed peak NO concentrations varied across the anatomical layers of the cerebellum, from 12 to 130 pm. The mean value found in the hippocampus was 200 pm. Much variation in the amplitudes recorded by individual detector cells was observed, this being attributable to their location at variable distances from the NO sources. From fits to the data, the NO concentrations at the source surfaces were 120 pm to 1.4 nm, and the underlying rates of NO generation were 36-350 nm/s, depending on area. Our measurements are 4-5 orders of magnitude lower than reported by some electrode recordings in cerebellum or hippocampus. In return, they establish coherence between the NO concentrations able to elicit physiological responses in target cells through guanylyl cyclase-linked NO receptors, the concentrations that neuronal NO synthase is predicted to generate locally, and the concentrations that neurons actually produce.     

Microbial community dynamics in a humic lake: differential persistence of common freshwater phylotypes

In an effort to better understand the factors contributing to patterns in freshwater bacterioplankton community composition and diversity, we coupled automated ribosomal intergenic spacer analysis (ARISA) to analysis of 16S ribosomal RNA (rRNA) gene sequences to follow the persistence patterns of 46 individual phylotypes over 3 years in Crystal Bog Lake. Additionally, we sought to identify linkages between the observed phylotype variations and known chemical and biological drivers. Sequencing of 16S rRNA genes obtained from the water column indicated the presence of phylotypes associated with the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, TM7 and Verrucomicrobia phyla, as well as phylotypes with unknown affiliation. Employment of the 16S rRNA gene/ARISA method revealed that specific phylotypes varied independently of the entire bacterial community dynamics. Actinobacteria, which were present on greater than 95% of sampling dates, did not share the large temporal variability of the other identified phyla. Examination of phylotype relative abundance patterns (inferred using ARISA fragment relative fluorescence) revealed a strong correlation between the dominant phytoplankton succession and the relative abundance patterns of the majority of individual phylotypes. Further analysis revealed covariation among unique phylotypes, which formed several distinct bacterial assemblages correlated with particular phytoplankton communities. These data indicate the existence of unique persistence patterns for different common freshwater phylotypes, which may be linked to the presence of dominant phytoplankton species.     

MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.     

Detecting microRNA activity from gene expression data

Background  

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions.     

The structural and electrochemical consequences of hydrogenating copper N2S2 Schiff base macrocycles

A series of cis and trans tetradentate copper macrocyclic complexes, of ring size 14-16, that employ amine and thioether donor groups are reported. Apart from 5,6,15,16-bisbenzo-8,13-diaza-1,4-dithia-cyclohexadecane copper(I) (cis-[Cu(H₄N_buS_en)]⁺) all of the complexes are obtained in the copper(II) form. Crystallographic analysis shows that the copper(II) complexes all adopt a distorted planar geometry around the copper. In contrast, cis-[Cu(H₄N_buS_en)]⁺ is found to adopt a distorted tetrahedral geometry. The complexes were subjected to electrochemical analysis in water and acetonitrile. The effect of the solvent, positions of the donor atoms (cis/trans) on E^1/2 is discussed as is the comparison of the electrochemical behaviour of these complexes with their parent Schiff base macrocycles.     

Identification of the molecular genetic basis of the low palmitic acid seed oil trait in soybean mutant line RG3 and association analysis of molecular markers with elevated seed stearic acid and reduced seed palmitic acid

The fatty acid composition of vegetable oil is becoming increasingly critical for its ultimate functionality and utilization in foods and industrial products. Partial chemical hydrogenation of soybean [Glycine max (L.) Merr.] oil increases oxidative stability and shelf life but also results in the introduction of trans fats as an unavoidable byproduct. Due to mandatory labeling of consumer products containing trans fats, conventional soybean oil has lost the ability to deliver the most appropriate economical functionality and oxidative stability, particularly for baking applications. Genetic improvement of the fatty acid profile of soybean oil is one method of meeting these new requirements for oil feedstocks. In this report, we characterized three mutant genetic loci controlling the saturated fatty acid content of soybean oil: two genes additively reduce palmitic acid content (fap1 and fap3-ug), and one gene independently elevates stearic acid content (fas). We identified a new null allele of fap3-ug/GmFATB1A (derived from line ELLP2) present in line RG3. The splicing defect mutation in a beta-ketoacyl-[acyl-carrier-protein] synthase III candidate gene located in the region mapped to fap1, derived originally from ethyl methane sulphonate mutant line C1726 (Cardinal et al. in Theor Appl Genet 127:97–111, 2014), was also present in line RG3. We also utilized the elevated stearic acid line RG7, which has previously been shown to contain novel mutant fas/SACPD-C alleles encoding stearoyl-acyl carrier protein desaturase (Boersma et al. in Crop Sci 52:1736–1742, 2012). Molecular marker assays have been developed to track these causative mutations and understand their contributions to seed oil fatty acid profiles in a recombinant inbred line population segregating for fap1, fap3-ug, and fas alleles.     

4E10-Resistant HIV-1 Isolated from Four Subjects with Rare Membrane-Proximal External Region Polymorphisms

Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope.