Field Application of a Rapid Spectrophotometric Method for Determination of Persulfate in Soil

Remediation of hydrocarbon contaminated soils can be performed both in situ and ex situ using chemical oxidants such as sodium persulfate. Standard methods for quantifying persulfate require either centrifugation or prolonged settling times. An optimized soil extraction procedure was developed for persulfate involving simple water extraction using a modified disposable syringe. This allows considerable saving of time and removes the need for centrifugation. The extraction time was reduced to only 5 min compared to 15 min for the standard approach. A comparison of the two approaches demonstrated that each provides comparable results. Comparisons were made using high (93 g kg⁻¹ soil) and low (9.3 g kg⁻¹ soil) additions of sodium persulfate to a petroleum hydrocarbon-contaminated soil, as well as sand spiked with diesel. Recoveries of 95±1% and 96±10% were observed with the higher application rate in the contaminated soil and spiked sand, respectively. Corresponding recoveries of 86±5% and 117±19% were measured for the lower application rate. Results were obtained in only 25 min and the method is well suited to batch analyses. In addition, it is suitable for application in a small field laboratory or even a mobile, vehicle-based system, as it requires minimal equipment and reagents.     

A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.     

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells: Role of Hypoxia Inducible Factor-1 and Wnt/Beta-Catenin Signaling

Background

Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective

We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods

Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results

At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion

Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.     

Latino racial choices: the effects of skin colour and discrimination on Latinos’ and Latinas’ racial self-identifications

Abstract

Are predictions that Hispanics will make up 25 per cent of the US population in 2050 reliable? The authors of this paper argue that these and other predictions are problematic insofar as they do not account for the volatile nature of Latino racial and ethnic identifications. In this light, the authors propose a theoretical framework that can be used to predict Latinos’ and Latinas’ racial choices. This framework is tested using two distinct datasets – the 1989 Latino National Political Survey and the 2002 National Survey of Latinos. The results from the analyses of both of these surveys lend credence to the authors’ claims that Latinas’ and Latinos’ skin colour and experiences of discrimination affect whether people from Latin America and their descendants who live in the US will choose to identify racially as black, white or Latina/o.     

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca²⁺ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His₁₀) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.     

EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation

Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.     

The Himalayas: Barrier and conduit for gene flow

The Himalayan mountain range is strategically located at the crossroads of the major cultural centers in Asia, the Middle East and Europe. Although previous Y‐chromosome studies indicate that the Himalayas served as a natural barrier for gene flow from the south to the Tibetan plateau, this region is believed to have played an important role as a corridor for human migrations between East and West Eurasia along the ancient Silk Road. To evaluate the effects of the Himalayan mountain range in shaping the maternal lineages of populations residing on either side of the cordillera, we analyzed mitochondrial DNA variation in 344 samples from three Nepalese collections (Newar, Kathmandu and Tamang) and a general population of Tibet. Our results revealed a predominantly East Asian‐specific component in Tibet and Tamang, whereas Newar and Kathmandu are both characterized by a combination of East and South Central Asian lineages. Interestingly, Newar and Kathmandu harbor several deep‐rooted Indian lineages, including M2, R5, and U2, whose coalescent times from this study (U2, >40 kya) and previous reports (M2 and R5, >50 kya) suggest that Nepal was inhabited during the initial peopling of South Central Asia. Comparisons with our previous Y‐chromosome data indicate sex‐biased migrations in Tamang and a founder effect and/or genetic drift in Tamang and Newar. Altogether, our results confirm that while the Himalayas acted as a geographic barrier for human movement from the Indian subcontinent to the Tibetan highland, it also served as a conduit for gene flow between Central and East Asia. Am J Phys Anthropol 151:169–182, 2013. © 2013 Wiley Periodicals, Inc.