A microfluidic processor for gene expression profiling of single human embryonic stem cells

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These cell population averaging data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies

Gene Splicing of an Invertebrate Beta Subunit (LCav?) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels

The accessory beta subunit (Cavβ) of calcium channels first appear in the same genome as Cav1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Cavβ subunits (β1, β2, β3, β4) which associate with four Cav1 channel isoforms (Cav1.1 to Cav1.4) and three Cav2 channel isoforms (Cav2.1 to Cav2.3). Here we assess the fundamentally-shared features of the Cavβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCav1, LCav2, and LCavβ). Invertebrate Cavβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Cav2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCavβ subunit. LCavβ will also slow the inactivation kinetics of LCav3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Cavβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCavβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Cav2.2 and Cavβ3 subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCav2 nor LCavβ) appears to be compatible with this observed property

Helium ion microscope – secondary ion mass spectrometry for geological materials

The helium ion microscope (HIM) is a focussed ion beam instrument with unprecedented spatial resolution for secondary electron imaging but has traditionally lacked microanalytical capabilities. With the addition of the secondary ion mass spectrometry (SIMS) attachment, the capabilities of the instrument have expanded to microanalysis of isotopes from Li up to hundreds of atomic mass units, effectively opening up the analysis of all natural and geological systems. However, the instrument has thus far been underutilised by the geosciences community, due in no small part to a lack of a thorough understanding of the quantitative capabilities of the instrument. Li represents an ideal element for an exploration of the instrument as a tool for geological samples, due to its importance for economic geology and a green economy, and the difficult nature of observing Li with traditional microanalytical techniques. Also Li represents a “best-case” scenario for isotopic measurements. Here we present details of sample preparation, instrument sensitivity, theoretical, and measured detection limits for both elemental and isotopic analysis as well as practicalities for geological sample analyses of Li alongside a discussion of potential geological use cases of the HIM–SIMS instrument

Comprehensive functional characterization of murine infantile Batten disease including Parkinson-like behavior and dopaminergic markers

Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is a neurodegenerative lysosomal storage disease caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). The PPT1-deficient mouse (Cln1−/−) is a useful phenocopy of human INCL. Cln1−/− mice display retinal dysfunction, seizures, motor deficits, and die at ~8 months of age. However, little is known about the cognitive and behavioral functions of Cln1−/− mice during disease progression. In the present study, younger (~1–2 months of age) Cln1−/− mice showed minor deficits in motor/sensorimotor functions while older (~5–6 months of age) Cln1−/− mice exhibited more severe impairments, including decreased locomotor activity, inferior cued water maze performance, decreased running wheel ability, and altered auditory cue conditioning. Unexpectedly, certain cognitive functions such as some learning and memory capabilities seemed intact in older Cln1−/− mice. Younger and older Cln1−/− mice presented with walking initiation defects, gait abnormalities, and slowed movements, which are analogous to some symptoms reported in INCL and parkinsonism. However, there was no evidence of alterations in dopaminergic markers in Cln1−/− mice. Results from this study demonstrate quantifiable changes in behavioral functions during progression of murine INCL and suggest that Parkinson-like motor/sensorimotor deficits in Cln1−/− mice are not mediated by dopamine deficiency

Employing Modular Polyketide Synthase Ketoreductases as Biocatalysts in the Preparative Chemoenzymatic Syntheses of Diketide Chiral Building Blocks

SummaryChiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, β-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100 mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale

The Hydrogen Epoch of Reionization Array Dish I: Beam Pattern Measurements and Science Implications

The Hydrogen Epoch of Reionization Array (HERA) is a radio interferometer aiming to detect the power spectrum of 21 cm fluctuations from neutral hydrogen from the Epoch of Reionization (EOR). Drawing on lessons from the Murchison Widefield Array (MWA) and the Precision Array for Probing the Epoch of Reionization (PAPER), HERA is a hexagonal array of large (14 m diameter) dishes with suspended dipole feeds. Not only does the dish determine overall sensitivity, it affects the observed frequency structure of foregrounds in the interferometer. This is the first of a series of four papers characterizing the frequency and angular response of the dish with simulations and measurements. We focus in this paper on the angular response (i.e., power pattern), which sets the relative weighting between sky regions of high and low delay, and thus, apparent source frequency structure. We measure the angular response at 137 MHz using the ORBCOMM beam mapping system of Neben et al. We measure a collecting area of 93 m^2 in the optimal dish/feed configuration, implying HERA-320 should detect the EOR power spectrum at z~9 with a signal-to-noise ratio of 12.7 using a foreground avoidance approach with a single season of observations, and 74.3 using a foreground subtraction approach. Lastly we study the impact of these beam measurements on the distribution of foregrounds in Fourier space.Comment: 13 pages, 9 figures. Replaced to match accepted ApJ versio

Early Childhood Nutrition Knowledge of Caregivers in Tanzania

Childhood stunting is a pressing health issue in Tanzania and results from chronic infections and inadequate nutrition. Educating caregivers on the nutritional determinants, their consequences, and appropriate solutions may improve nutrition-related practices among caregivers in Tanzania. The purpose of this study was to identify factors associated with Tanzanian caregivers’ knowledge of childhood nutrition practices. Data for this study came from a cross-sectional survey of 4,095 caregivers of children under 24 months living in the Geita, Kagera, Kigoma, Mwanza, and Shinyanga regions of Tanzania. Complete responses relating to demographic and socioeconomic factors, media exposure, and early childhood nutrition knowledge were analyzed using multiple linear regression modeling techniques. Caregivers’ knowledge concerning proper early childhood nutrition practices was found to be significantly related to using a mobile banking account (p<.0001), owning a working radio with batteries (p<.0001), having watched television recently (p<.0001), residing in a southern lake region (p<.0001), affiliating with a Christian faith (p=0.0027), having more children under the age of 5 (p=0.0005), having received advice on maternal nutrition before pregnancy (p<.0001) and having received advice from a community health worker (p=0.0184). Living in a rural environment (p<.0001) and speaking a non-mainstream language (p<0.05) were significantly associated with decreased knowledge. The influences of media and technology, socio-demographic factors and traditional health education may be important in the development of accurate childhood nutrition knowledge among caregivers. These factors may be targeted for future community health worker efforts with vulnerable populations in Tanzania to prevent stunting

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines.

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo

Radiocarbon dating and cultural dynamics across Mongolia’s early pastoral transition

All necessary permits were obtained for the described study, which complied with all relevant regulations. Collaboration contract between the Max Planck Institute for the Science of Human HIstory and the National University of Mongolia began on the 10th November, 2016. Export number 10/413 (7b/52) was received on the 2nd Feb, 2017 (#A0109258, MN DE 7 643). This research was supported by the Max Planck Institute for the Science of Human History. Special thanks to Dr. Katerina Douka and the Oxford Radiocarbon Accelerator Laboratory for conducting 14C analysis, and to all of the original excavators and authors who published the radiocarbon dates cited in this study.Peer reviewedPublisher PD