The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities

Optical microscopy of single bacteria growing on solid agarose support is a powerful method for studying the natural heterogeneity in growth and gene expression. While the material properties of agarose make it an excellent substrate for such studies, the sheer number of exponentially growing cells eventually overwhelms the agarose pad, which fundamentally limits the duration and the throughput of measurements. Here we overcome the limitations of exponential growth by patterning agarose pads on the sub-micron-scale. Linear tracks constrain the growth of bacteria into a high density array of linear micro-colonies. Buffer flow through microfluidic lines washes away excess cells and delivers fresh nutrient buffer. Densely patterned tracks allow us to cultivate and image hundreds of thousands of cells on a single agarose pad over 30-40 generations, which drastically increases single-cell measurement throughput. In addition, we show that patterned agarose can facilitate single-cell measurements within bacterial communities. As a proof-of-principle, we study a community of E. coli auxotrophs that can complement the amino acid deficiencies of one another. We find that the growth rate of colonies of one strain decreases sharply with the distance to colonies of the complementary strain over distances of only a few cell lengths. Because patterned agarose pads maintain cells in a chemostatic environment in which every cell can be imaged, we term our device the single-cell chemostat. High-throughput measurements of single cells growing chemostatically should greatly facilitate the study of a variety of microbial behaviours.Molecular and Cellular Biolog

Author Correction: Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy

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The linked survival prospects of siblings : evidence for the Indian states

This paper reports an analysis of micro-data for India that shows a high correlation in infant mortality among siblings. In 13 of 15 states, we identify a causal effect of infant death on the risk of infant death of the subsequent sibling (a scarring effect), after controlling for mother-level heterogeneity. The scarring effects are large, the only other covariate with a similarly large effect being mother’s (secondary or higher) education. The two states in which evidence of scarring is weak are Punjab, the richest, and Kerala, the socially most progressive. The size of the scarring effect depends upon the sex of the previous child in three states, in a direction consistent with son-preference. Evidence of scarring implies that policies targeted at reducing infant mortality will have social multiplier effects by helping avoid the death of subsequent siblings. Comparison of other covariate effects across the states offers some interesting new insights

Robust Circadian Oscillations in Growing Cyanobacteria Require Transcriptional Feedback

The remarkably stable circadian oscillations of single cyanobacteria enable a population of growing cells to maintain synchrony for weeks. The cyanobacterial pacemaker is a posttranslational regulation (PTR) circuit that generates circadian oscillations in the phosphorylation state of the clock protein KaiC. Layered on top of the PTR is transcriptional-translational feedback regulation (TTR), common to all circadian systems, consisting of a negative feedback loop in which KaiC regulates its own production. We found that the PTR circuit is sufficient to generate oscillations in growing cyanobacteria. However, in the absence of TTR, individual oscillators were less stable and synchrony was not maintained in a population of cells. Experimentally constrained mathematical modeling reproduced sustained oscillations in the PTR circuit alone and demonstrated the importance of TTR for oscillator synchrony.Chemistry and Chemical BiologyMolecular and Cellular BiologyPhysic

Chandra and Very Large Array Observations of the Nearby Sd Galaxy NGC 45

We present an analysis of high angular resolution observations made in the X-ray and the radio with the Chandra X-ray Observatory and the Karl Jansky Very Large Array (VLA), respectively, of the nearby spiral galaxy NGC 45. This galaxy is the third that we have considered in a study of the supernova remnant (SNR) populations of nearby spiral galaxies and the present work represents the first detailed analysis of the discrete X-ray and radio source populations of this galaxy. We analyzed data sets from the three pointed observations made of this galaxy with Chandra along with a merged data set obtained from combining these data sets: the total effective exposure time of the merged data set is 63515 s. A total of 25 discrete X-ray sources are found in the entire field of view of the ACIS-S3 chip, with 16 sources found within the visual extent of the galaxy. We estimate that as many as half of the sources detected in the entire field of view of the ACIS-S3 chip and seven of the sources detected in the optical extent of NGC 45 may be background sources. We analyzed the spectral properties of the discrete X-ray sources within the galaxy and conclude that the majority of these sources are X-ray binaries. We have searched for counterparts at different wavelengths to the discrete X-ray sources and we find two associations: one with a star cluster and the other with a background galaxy. We have found one source that is clearly variable within one observation and seven that are seen to vary from one observation to another. We also conduct a photometric analysis to determine the near-infrared fluxes of the discrete X-ray sources in Spitzer Infrared Array Camera channels. We constructed a cumulative luminosity function of the discrete X-ray sources seen toward NGC 45: taking into account simultaneously the luminosity function of background sources, the fitted slope of the cumulative luminosity function Γ = –1.3_(-1.6)^(+0.7) (all error bounds correspond to 90% confidence intervals). The VLA observations reveal seven discrete radio sources: we find no overlaps between these sources and the X-ray detected sources. Based on their measured spectral indices and their locations with respect to the visible extent of NGC 45, we classify one source as a candidate radio SNR associated with the galaxy and the others as likely background galaxies seen in projection toward NGC 45. Finally, we discuss the properties of a background cluster of galaxies (denoted as CXOU J001354.2–231254.7) seen in projection toward NGC 45 and detected by the Chandra observations. The fit parameters to the extracted Chandra spectra of this cluster are a column density N_H = 0.07(<0.14) × 10^(22) cm^(−2), a temperature kT = 4.22_(-1.42)^(+2.08) keV, an abundance Z = 0.30(<0.75) relative to solar and a redshift z = 0.28 ± 0.14. From the fit parameters we derive an electron number density n_e = 4(±1) × 10^(−3) cm^(−3), an unabsorbed X-ray luminosity L_(0.5-7.0keV) ~ 8.77(±0.96) × 10^(43) erg s^(−1) for the cluster and an X-ray emitting mass M = 2.32(±1.75) × 10^(12)M_☉

Statistical Analysis of Molecular Signal Recording

A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.United States. Defense Advanced Research Projects Agency. Living Foundries ProgramGoogle (Firm)New York Stem Cell Foundation. Robertson Neuroscience Investigator AwardNational Institutes of Health (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Transformative R01 1R01GM104948)National Institutes of Health (U.S.) (Single Cell Grant 1 R01 EY023173)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (CAREER Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS1042134)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution

Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma

SummaryWe describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC