A Rapid and Quantitative Assay to Estimate Gene Transfer into Retrovirally Transduced Hematopoietic Stem/Progenitor Cells Using a 96-Well Format PCR and Fluorescent Detection System Universal for MMLV-Based Proviruses

Overview summary The polymerase chain reaction (PCR) analysis of colonies of clonogenic cells growing in methylcellulose medium is a routine procedure to estimate the frequency of retroviral transduction into hematopoietic stem/progenitor cells. This study describes a sensitive assay system that takes advantage of the standard 96-well format to expedite the processing of single methylcellulose colonies. Assay sensitivity is dependent on a PCR primer pair which amplifies a region of the ψ packaging sequence of all Moloney-based retroviruses tested. Using this primer pair, we present the optimized PCR conditions for the analysis of single colonies of clonogenic cells growing in methylcellulose medium as well as the conditions for a semiquantitative bulk PCR assay to estimate the transduction frequency immediately following the transduction protocol. This PCR primer pair, along with the capability for more rapid screening of hematopoietic stem/progenitor colonies, is especially useful for the laboratory that is screening a number of different retroviral constructions for their transduction efficiency.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63170/1/hum.1996.7.3-343.pd

ANNUAL REPORT 2022 - Data collection and analysis in support of single and multispecies stock assessments in Chesapeake Bay: The Chesapeake Bay Multispecies Monitoring and Assessment Program

The threats affecting living marine resources are diverse, including overfishing, climate change, and pollution. In response to long-term challenges in fisheries management, a more holistic evaluation of the natural and anthropogenic drivers of populations sizes is needed. Ecosystem management (EM), a suite of strategies that incorporate ecosystem considerations into fisheries and ecosystem management, can be difficult to implement in practice. In the Mid-Atlantic, efforts to implement EM are ongoing; one output has been the annual ‘State of the Ecosystem Report’ for the region, synthesizes available data on a variety of environmental, ecological, and socioeconomic factors. Historically, the data needed for EM in Chesapeake Bay were either incomplete or nonexistent. In 2002, the Chesapeake Bay Multispecies Monitoring and Assessment Program (ChesMMAP) was developed to assist in filling these data gaps, and ultimately to support Bay-specific species and ecosystem assessment modeling. ChesMMAP is a fishery-independent monitoring survey that uses a bottom trawl designed to sample late juvenile-to-adult fishes in the mainstem of Chesapeake Bay. Since 2002, this program has provided data on relative abundance, length, weight, sex ratio, maturity, age, and trophic interactions for several important fish species that inhabit the Bay seasonally. In this annual progress report, we synthesize available biological data on 12 bony fishes that support local recreational fisheries, including abundance (biomass and number), length- and age-structure, sex ratio, maturity stage, and diet composition. However, in 2019, the survey underwent a major redesign: VIMS took possession of a new research vessel, the R/V Virginia, and the survey bottom trawl gear was replaced with a net consistent with the gear used by other regional bottom trawl surveys (i.e., the Northeast Area Monitoring and Assessment Program and Northeast Fisheries Science Center trawl surveys). At this time, the survey stratification was revised and changes were made to the cruise schedule. During 15 calibration cruises (2019-2022), 516 paired-tows were completed, which was deemed sufficient for robust statistical analysis. Species-specific intercalibrations have been conducted by applying log-Gaussian Cox processes to the paired-tow data and modeling the size distribution of the population at each sampling site and the size-structured clustering of fish at small temporal and spatial scales. The manuscript describing the application to the ChesMMAP calibration data is currently in review. The ChesMMAP data inform Bay- and coast-wide fisheries management decisions and the broader use of these data in theses, dissertations, and the peer-reviewed literature contributes to a better understanding of the Bay ecosystem

Spectral Variations of Of?p Oblique Magnetic Rotator Candidates in the Magellanic Clouds

Optical spectroscopic monitoring has been conducted of two O stars in the Small and one in the Large Magellanic Cloud, the spectral characteristics of which place them in the Of?p category, which has been established in the Galaxy to consist of oblique magnetic rotators. All of these Magellanic stars show systematic spectral variations typical of the Of?p class, further strengthening their magnetic candidacy to the point of virtual certainty. The spectral variations are related to photometric variations derived from OGLE data by Naze et al. (2015) in a parallel study, which yields rotational periods for two of them. Now circular spectropolarimetry is required to measure their fields, and ultraviolet spectroscopy to further characterize their low-metallicity, magnetically confined winds, in support of hydrodynamical analyses.Comment: 18 pages, 6 figures, accepted for publication by A

Radio Emission and Orbital Motion from the Close-encounter Star–Brown Dwarf Binary WISE J072003.20–084651.2

We report the detection of radio emission and orbital motion from the nearby star–brown dwarf binary WISE J072003.20–084651.2AB. Radio observations across the 4.5–6.5 GHz band with the Very Large Array identify at the position of the system quiescent emission with a flux density of 15 ± 3 μJy, and a highly polarized radio source that underwent a 2–3 minute burst with peak flux density 300 ± 90 μJy. The latter emission is likely a low-level magnetic flare similar to optical flares previously observed for this source. No outbursts were detected in separate narrow-band Hα monitoring observations. We report new high-resolution imaging and spectroscopic observations that confirm the presence of a co-moving T5.5 secondary and provide the first indications of three-dimensional orbital motion. We used these data to revise our estimates for the orbital period (4.1_(-1.3)^(+2.7) year) and tightly constrain the orbital inclination to be nearly edge-on (93º6^(+1º6)_(−1º4)), although robust measures of the component and system masses will require further monitoring. The inferred orbital motion does not change the high likelihood that this radio-emitting very low-mass binary made a close pass to the Sun in the past 100 kyr

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices

The FIRST-Optical-VLA Survey for Lensed Radio Lobes

We present results from a survey for gravitationally lensed radio lobes. Lensed lobes are a potentially richer source of information about galaxy mass distributions than lensed point sources, which have been the exclusive focus of other recent surveys. Our approach is to identify radio lobes in the FIRST catalog and then search optical catalogs for coincident foreground galaxies, which are candidate lensing galaxies. We then obtain higher-resolution images of these targets at both optical and radio wavelengths, and obtain optical spectra for the most promising candidates. We present maps of several radio lobes that are nearly coincident with galaxies. We have not found any new and unambiguous cases of gravitational lensing. One radio lobe in particular, FOV J0743+1553, has two hot spots that could be multiple images produced by a z=0.19 spiral galaxy, but the lensing interpretation is problematic.Comment: 38 pages, 18 figures, aastex, accepted to A

Long-term site fidelity of endangered smalltooth sawfish (Pristis pectinata) from different mothers

Understanding how endangered species use nursery habitats is vital for recovery planning. Research on the smalltooth sawfish (Pristis pectinata) has shown that areas of estuarine nurseries, called hotspots, are used consistently. The objectives of our study were 1) to determine whether 10 young-of-the-year smalltooth sawfish in an artificial, non-main-stem portion (i.e., a seawall canal system) of a hotspot were descended from one or different mothers and 2) to document long-term habitat use by these individuals. At least 4 mothers contributed to the group, which comprised siblings, half-siblings, and unrelated individuals. Young sawfish exhibited site fidelity to their capture location, spending 61% of their time there. Continuous residency lasted as long as 86 days, but these fish made small-scale diel

Hybrid Inflatable Pressure Vessel

Figure 1 shows a prototype of a large pressure vessel under development for eventual use as a habitable module for long spaceflight (e.g., for transporting humans to Mars). The vessel is a hybrid that comprises an inflatable shell attached to a rigid central structural core. The inflatable shell is, itself, a hybrid that comprises (1) a pressure bladder restrained against expansion by (2) a web of straps made from high-strength polymeric fabrics. On Earth, pressure vessels like this could be used, for example, as portable habitats that could be set up quickly in remote locations, portable hyperbaric chambers for treatment of decompression sickness, or flotation devices for offshore platforms. In addition, some aspects of the design of the fabric straps could be adapted to such other items as lifting straps, parachute straps, and automotive safety belts. Figure 2 depicts selected aspects of the design of a vessel of this type with a toroidal configuration. The bladder serves as an impermeable layer to keep air within the pressure vessel and, for this purpose, is sealed to the central structural core. The web includes longitudinal and circumferential straps. To help maintain the proper shape upon inflation after storage, longitudinal and circumferential straps are indexed together at several of their intersections. Because the web is not required to provide a pressure seal and the bladder is not required to sustain structural loads, the bladder and the web can be optimized for their respective functions. Thus, the bladder can be sealed directly to the rigid core without having to include the web in the seal substructure, and the web can be designed for strength. The ends of the longitudinal straps are attached to the ends of the rigid structural core by means of clevises. Each clevis pin is surrounded by a roller, around which a longitudinal strap is wrapped to form a lap seam with itself. The roller is of a large diameter chosen to reduce bending of the fibers in the strap. The roller also serves to equalize the load in the portions of the strap on both sides of the clevis pin. The lap seam is formed near the clevis by use of a tapered diamond stitch: This stitch is designed specifically to allow fibers in the stitch and strap to relax under load in such a manner that the load becomes more nearly evenly distributed among all fibers in the stitch region. Thus, the tapered diamond stitch prevents load concentrations that could cause premature failure of the strap and thereby increases the strength of the strap/structural-core joint. The lap seam can be rated at >90 percent of the strength of the strap material

Nephrogenic systemic fibrosis associated with stromal and vascular calcification, report of two cases

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75035/1/j.1600-0560.2008.01205.x.pd