From Genes to Ambiguity: A Case Study Exploring the Enigmatic Connection Between Chromosome 13q Deletion Syndrome and Ambiguous Genitalia.

During development, the deletion of DNA from chromosome 13's short arm (q) causes a chromosomal abnormality known as chromosome 13q deletion syndrome. Chromosome 13 terminal deletions are rare and may cause various congenital disabilities, and only a few cases have been reported in the literature. The extent of chromosome 13q deletion syndrome changes lacks consistent clinical features, with no recorded cases of genital ambiguity until now. We report the case of a newborn male patient whose testes had descended on both sides; he had ambiguous genitalia, and the dorsal surface of his penis was attached to his scrotal sac. An abnormal karyotype (46, XY, deletion (13) q33) was discovered by using a G-banding analysis of chromosomes in a blood sample taken from the periphery, which revealed a deletion of chromosome 13 at the end of the first 10 cells. We can better characterize chromosome 13q deletions by establishing stronger correlations between karyotype and the distinctive phenotypes of haploinsufficient genes found on the chromosome.

Longer-term temporal trends in PFAS concentrations in Midwestern landfill leachate.

It is well documented that per- and polyfluoroalkyl substances (PFAS) are routinely detected in leachate from municipal solid waste (MSW) landfills. PFAS-containing products are ubiquitous in society and in end-of-life discarded materials. While considerable data have been generated in recent years for PFAS concentrations in landfill leachate, little published information exists concerning longer-term trends in leachate concentrations. In this study, we examine a ten-year dataset for three MSW landfills located in the upper Midwestern United States where leachate PFAS testing has been performed on a quarterly basis. The significance of these data are considered in light of phase-out initiatives implemented in the US manufacturing companies for longer-chain PFAS including perfluorooctanoic acid (PFOA), perfluorooctane sulphonate (PFOS) and perfluorononanoic acid (PFNA). Findings from this study indicate that the average concentration of longer-chain PFOA in the three landfills decreased from 2400 to 1900 ng/L and the average PFOS concentrations decreased from 1000 to 370 ng/L. This may be indicative of reduced domestic production and use of these compounds. The phase-out of long-chain PFAS has also led to the introduction of shorter-chain replacement compounds such as perfluorobutanoic acid, perfluorobutane sulphonate and perfluorohexanoic acid, and this paper also examines long-term trends in leachate concentrations of these compounds.

Modeling methane oxidation in landfill cover soils as indicator of functional stability with respect to gas management.

A performance-based method for evaluating methane (CH4) oxidation as the best available control technology (BACT) for passive management of landfill gas (LFG) was applied at a municipal solid waste (MSW) landfill in central Washington, USA, to predict when conditions for functional stability with respect to LFG management would be expected. The permitted final cover design at the subject landfill is an all-soil evapotranspirative (ET) cover system. Using a model, a correlation between CH4 loading flux and oxidation was developed for the specific ET cover design. Under Washington's regulations, a MSW landfill is functionally stable when it does not present a threat to human health or the environment (HHE) at the relevant point of exposure (POE), which was conservatively established as the cover surface. Approaches for modeling LFG migration and CH4 oxidation are discussed, along with comparisons between CH4 oxidation and biodegradation of non-CH4 organic compounds (NMOCs). The modeled oxidation capacity of the ET cover design is 15 g/m2/day under average climatic conditions at the site, with 100% oxidation expected on an annual average basis for fluxes up to 8 g/m2/day. This translates to a sitewide CH4 generation rate of about 260 m3/hr, which represents the functional stability target for allowing transition to cover oxidation as the BACT (subject to completion of a confirmation monitoring program). It is recognized that less than 100% oxidation might occur periodically if climate and/or cover conditions do not precisely match the model, but that residual emissions during such events would be de minimis in comparison with published limit values. Accordingly, it is also noted that nonzero net emissions may not represent a threat to HHE at a POE (i.e., a target flux between 8 and 15 g/m2/day might be appropriate for functional stability) depending on the site reuse plan and distance to potential receptors.Implications: This study provides a scientifically defensible method for estimating when methane oxidation in landfill cover soils may represent the best available control technology for residual landfill gas (LFG) emissions. This should help operators and regulators agree on the process of safely eliminating active LFG controls in favor of passive control measures once LFG generation exhibits asymptotic trend behavior below the oxidation capacity of the soil. It also helps illustrate the potential benefits of evolving landfill designs to include all-soil vegetated evapotranspirative (ET) covers that meet sustainability objectives as well as regulatory performance objectives for infiltration control.

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

Modeling the effects of vegetation on methane oxidation and emissions through soil landfill final covers across different climates.

Plant roots are reported to enhance the aeration of soil by creating secondary macropores which improve the diffusion of oxygen into soil as well as the supply of methane to bacteria. Therefore, methane oxidation can be improved considerably by the soil structuring processes of vegetation, along with the increase of organic biomass in the soil associated with plant roots. This study consisted of using a numerical model that combines flow of water and heat with gas transport and oxidation in soils, to simulate methane emission and oxidation through simulated vegetated and non-vegetated landfill covers under different climatic conditions. Different simulations were performed using different methane loading flux (5-200 g m(-2) d(-1)) as the bottom boundary. The lowest modeled surface emissions were always obtained with vegetated soil covers for all simulated climates. The largest differences in simulated surface emissions between the vegetated and non-vegetated scenarios occur during the growing season. Higher average yearly percent oxidation was obtained in simulations with vegetated soil covers as compared to non-vegetated scenario. The modeled effects of vegetation on methane surface emissions and percent oxidation were attributed to two separate mechanisms: (1) increase in methane oxidation associated with the change of the physical properties of the upper vegetative layer and (2) increase in organic matter associated with vegetated soil layers. Finally, correlations between percent oxidation and methane loading into simulated vegetated and non-vegetated covers were proposed to allow decision makers to compare vegetated versus non-vegetated soil landfill covers. These results were obtained using a modeling study with several simplifying assumptions that do not capture the complexities of vegetated soils under field conditions.

Mastering translational medicine: interdisciplinary education for a new generation.

Graduate-level education in translational medicine will require more than just scientific research.

Evaluation of the efficiency of an experimental biocover to reduce BTEX emissions from landfill biogas.

Landfill emissions include volatile organic compounds (VOCs) and, particularly, benzene, toluene, ethyl-benzene and xylene isomers (collectively called BTEX). The latter are the most common VOCs found in landfill biogas. BTEX affect air quality and may be harmful to human health. In conjunction with a study aiming to evaluate the efficiency of passive methane oxidizing biocovers, a complementary project was developed with the specific goal of evaluating the reduction in VOC emissions due to the installation of a biocover. One of the biocovers constructed at the Saint-Nicéphore (Quebec, Canada) landfill site was instrumented for this purpose. The total BTEX concentration in the raw biogas ranged from 28.7 to 65.4ppmv, and the measured concentration of BTEX in biogas emitted through the biocover ranged from below the limit of detection (BLD) to 2.1ppmv. The other volatile organic compounds (OVOCs) concentration varied from 18.8 to 40.4ppmv and from 0.8 to 1.2ppmv in the raw biogas and in the emitted biogas, respectively. The results obtained showed that the biocover effectiveness ranged from 67% to 100% and from 96% to 97% for BTEX and OVOC, respectively.

Community-generated recommendations regarding the urban nutrition and tobacco environments: a photo-elicitation study in Philadelphia.

INTRODUCTION: Overweight, obesity, and tobacco use are major preventable causes of disability, disease, and death. In 2010, 25% of Philadelphia adults smoked, and 66% were overweight or obese. To address these health threats, the Philadelphia Department of Public Health launched Get Healthy Philly, an initiative to improve the city's nutrition, physical activity, and tobacco environments. The objective of this assessment was to identify residents' perspectives on threats to health and opportunities for change in the local food and tobacco environments. METHODS: Participants (N = 48) took photographs to document their concerns regarding Philadelphia's food and tobacco environments and participated in photo-elicitation interviews. We coded photographs and interview transcripts and identified key themes. RESULTS: Participants proposed interventions for nutrition 4 times more often than for tobacco. Participants spontaneously articulated the need for multilevel change consistent with the ecological model of health behavior, including changes to policies (food assistance program provisions to encourage healthful purchases), local and school environments (more healthful corner store inventories and school meals), and individual knowledge and behavior (healthier food purchases). Participants often required interviewer prompting to discuss tobacco, and they suggested interventions including changes in advertising (a local environmental concern) and cigarette taxes (a policy concern). CONCLUSION: Participants were well versed in the relevance to health of nutrition and physical activity and the need for multilevel interventions. Their responses suggested community readiness for change. In contrast, participants' more limited comments regarding tobacco suggested that prevention and control of tobacco use were perceived as less salient public health concerns.

Absorption, distribution, metabolism, and excretion of [1-¹4C]-perfluorohexanoate ([¹4C]-PFHx) in rats and mice.

The absorption, tissue distribution, elimination, and metabolism of [1-¹4C]-PFHx in rats and mice dosed orally at 2 or 100 mg/kg was evaluated following a single dose or after 14 consecutive doses. Absorption was rapid in rats as evidenced by a short time to maximum concentration (C(max)) of 30 min in male rats and 15 min in female rats at both the 2 and 100mg/kg dose level. The plasma elimination half-life was somewhat longer in males (1.5-1.7 h) than in females (0.5-0.7 h). Absorption in the mouse was also rapid with the maximum plasma concentration occurring between 15 and 30 min after dosing. The maximum concentration was not appreciably different between male and female mice (8 µg equiv./g at 2 mg/kg; ~350 µg equiv./g at 100 mg/kg). The primary route of elimination was via the urine. PFHx was not metabolized in rat or mouse hepatocytes, nor were any metabolites observed after oral dosing in either rodent species. Essentially 100% of the dose was eliminated in urine within 24 h demonstrating that PFHx is readily absorbed and bioavailability approaches 100%, even at a dose as high as 100 mg/kg. The route and extent of elimination was unchanged after 14 days of daily dosing. Tissues were collected at three time points (rat: 0.5, 2, and 24 h; mice: 0.25, 1, and 24 h) after dosing to investigate the tissue clearance kinetics of PFHx following a single dose at 2 or 100 mg/kg. In all tissues except skin, PFHx was not quantifiable 24 h after dosing in both sexes of the two species.

Performance of a full-scale hydrogen-storage tank based on complex hydrides.

Designing and building a full scale hydrogen storage system revealed several engineering challenges and also demonstrated the capabilities of complex hydrides. Three kg of hydrogen was stored in a four module system using modified sodium alanate as the storage media. Extensive testing of this system demonstrated the ability to follow aggressive hydrogen demand schedules that simulate actual driving. Extensive use of detailed models greatly improved the design and eventual performance of the storage system; the test data permitted further refinement of the models.

Microfluidic environment for high density hepatocyte culture.

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm(2)) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 microl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.

Inducible expression of a degradation-resistant form of p27Kip1 causes growth arrest and apoptosis in breast cancer cells.

The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E-CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines overexpressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.

A comprehensive assessment of p53-responsive genes following adenoviral-p53 gene transfer in Bcl-2-expressing prostate cancer cells.

The p53 protein can induce cell cycle arrest or apoptosis following activation in response to DNA damage. The function of p53 is largely mediated by regulating the expression of downstream target genes. Adenoviral-p53 gene transfer (Ad-p53) is currently being evaluated in clinical trials as a therapeutic intervention. Tumor response is likely to be influenced by context-dependent variables, such as expression of bcl-2. Bcl-2 is upregulated in a variety of neoplasms, and can inhibit p53-dependent apoptosis. It was therefore of interest to use a global genomic strategy to assess gene expression following Ad-p53 gene transfer and to determine if the expression of specific Ad-p53-responsive genes could be modulated in the context of bcl-2 gene deregulation. cDNA arrays were used to identify p53-responsive genes following Ad-p53 gene transfer in control and bcl-2-overexpressing PC3 prostate cancer cells. A total of 40 transcripts were significantly upregulated by Ad-p53 in both control and bcl-2-transfectant PC3 cells. Conversely, 19 transcripts were significantly repressed in both cell lines. These Ad-p53-responsive transcripts included previously identified p53 targets, known genes representing candidate p53 targets, and transcripts identified as expressed sequence tags. A subset of 15 transcripts was differentially modulated by Ad-p53 in the context of bcl-2. Some of these genes were also differentially modulated in LNCaP (wt p53) cells following DNA damage. These results document a number of potential p53 targets and mediators of therapeutically relevant genotoxic stress. The findings further suggest that bcl-2 may inhibit cell death at multiple points downstream of p53 activation.

Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses.

A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection approximately 10-100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity approximately 5-50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi-gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi-gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.

A mammalian cell regulatory agent, CeReS-18, inhibits yeast cell proliferation but not bacterial replication.

A cell regulatory sialoglycopeptide, CeReS-18, purified from intact bovine cerebral cortex cells, has exhibited the capability of reversibly inhibiting cellular DNA synthesis and the proliferation of a wide array of mammalian cells. In the present study, the effect of CeReS-18 on the proliferation of bacterial ( Bacillus cereus and Escherichia coli) and yeast ( Saccharomyces cerevisiae and Schizosaccharomyces pombe) cells was investigated. The results showed that replication and viability of the bacterial cells were not affected by CeReS-18 at any concentration tested, including 15-fold higher than that used for inhibiting mouse 3T3 cell proliferation. In contrast to bacterial cells, CeReS-18 was able to inhibit the replication of yeast cells, in a concentration-dependent, reversible manner, and the addition of calcium to the culture medium could abrogate the inhibitory effect of CeReS-18. A cytotoxic effect of CeReS-18 on both yeast cell species was observed when it was applied at higher concentrations.

A cell regulatory agent, CeReS-18, inhibits mouse 3T6 cell proliferation but not polyomavirus replication.

We have purified a cell regulatory sialoglycopeptide, CeReS-18, from intact bovine cerebral cortex cells. This is an 18-kDa molecule that reversibly inhibits cellular DNA synthesis and the proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on mouse 3T6 host cell proliferation and polyomavirus replication was investigated. The results showed that CeReS-18 was able to inhibit 3T6 cell cycling in a concentration-dependent, calcium-sensitive, and reversible manner. Despite the inhibition of cell proliferation, CeReS-18 did not influence polyomavirus infection of 3T6 cells. Indirect immunofluorescent assays revealed that CeReS-18-treated, and cell cycle-arrested, 3T6 cells remained permissive to polyomavirus replication. Electron microscopy and immunogold labeling showed that new viral particles were assembled inside the nuclei of infected cells in the presence of CeReS-18 and during cell cycle arrest. The cellular requirements for the replication of polyomavirus DNA and the synthesis of viral proteins, as well as for the assembly of viral particles, therefore, remained available in CeReS-18-inhibited 3T6 cells. In addition, although polyomavirus infection can be mitogenic, infection of CeReS-18-treated 3T6 cells did not reverse the cell cycle arrest mediated by this cell cycle inhibitor.