A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity

Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 106 variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks

Assessment of Vessel Density on Non-Contrast Computed Tomography to Detect Basilar Artery Occlusion

Introduction: Basilar artery occlusion (BAO) may be clinically occult due to variable and non-specific symptomatology. We evaluated the qualitative and quantitative determination of a hyperdense basilar artery (HDBA) on non-contrast computed tomography (NCCT) brain for the diagnosis of BAO.Methods: We conducted a case control study of patients with confirmed acute BAO vs a control group of suspected acute stroke patients without BAO. Two EM attending physicians, one third-year EM resident, and one medical student performed qualitative and quantitative assessments for the presence of a HDBA on axial NCCT images. Our primary outcome measures were sensitivity and specificity for BAO. Our secondary outcomes were inter-rater and intra-rater reliability of the qualitative and quantitative assessments.Results: We included 60 BAO and 65 control patients in our analysis. Qualitative assessment of the hyperdense basilar artery sign was poorly sensitive (54%–72%) and specific (55%–89%). Quantitative measurement improved the specificity of hyperdense basilar artery assessment for diagnosing BAO, with a threshold of 61.0–63.8 Hounsfield units demonstrating relatively high specificity of 85%–94%. There was moderate inter-rater agreement for the qualitative assessment of HDBA (Fleiss’ kappa statistic 0.508, 95% confidence interval: 0.435–0.581). Agreement improved for quantitative assessments, but still fell in the moderate range (Shrout-Fleiss intraclass correlation coefficient: 0.635). Intra-rater reliability for the quantitative assessments of the two attending physician reviewers demonstrated substantial consistency.Conclusion: Our results highlight the importance of carefully examining basilar artery density when interpreting the NCCT of patients with altered consciousness or other signs and symptoms concerning for an acute basilar artery occlusion. If the Hounsfield unit density of the basilar artery exceeds 61 Hounsfield units, BAO should be highly suspected

Aberrant Development of Thymocytes in Mice Lacking Laminin-2

In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin- 2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4-CD8-) and SP (CD4+CD8-, CD4-CD8+) populations and a significant decrease in the DP (CD4+CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice

Visualization of defects in single-crystal and thin-film PdCoO2 using aberration-corrected scanning transmission electron microscopy

Funding: This work was primarily supported by the U.S. Department of Energy, Office of Basic Sciences, Division of Materials Sciences and Engineering, under Award No. DE-SC0002334.Single-crystal delafossite PdCoO2 is known to have an extremely low intrinsic impurity concentration of ~0.001%, demonstrating extraordinarily high conductivity with a mean free path of ~20 microns at low temperatures. However, when grown as thin films, the resistivity at room temperature increases by a factor of 3 to 80 times, depending on the film thickness. Using scanning transmission electron microscopy, we identify different classes of defects for the single crystal vs epitaxial thin film. The dominant defect for single-crystal PdCoO2 is found to be ribbon-like defects. For the thin films, we identify different types of defects arising in epitaxial thin films mainly due to substrate termination that disrupt the lateral connectivity of the conducting planes. Our results are consistent with the high conductivity of single crystals and increased electrical resistivity of the thin films compared to that of single crystals, suggesting that selecting a proper substrate, improving surface quality, and reducing the step density are the keys to enhance the film quality for utilizing PdCoO2 as a platform for future applications.PostprintPeer reviewe

Extractabilidade de metais pesados em um solo tratado com lodo de esgoto

Few investigations have been carried out about the comparison of desorption rate and amount of heavy metals extracted successively by organic acid mixtures mimicking the rhizosphere and routine extractants in sewage sludge-amended soils. Extractions of Zn, Cd, Ni, Cr, and Cu were performed in samples of a sewage sludge-amended soil using seven extractants: four organic acid mixtures and three routine extractants (DTPA, Mehlich-I, and ammonium acetate). Results from single pass extractions, in which the extractable metal contents were determined by simply extracting the soil a single time, as well as from 15 successive extractions, in which the solid residues of the first extraction was successively extracted 14 additional times, of heavy metals were analyzed. The extractability of heavy metals in a single pass extraction was, in general, as follows: Mehlich-I >; DTPA >; organic acids >; NH4OAc. The highest rates of extraction followed the general order: DTPA >; Mehlich-I >; organic acids >; NH4OAc. While Mehlich-I presented the highest extractability of heavy metals among studied extractants, DTPA showed a high extractability of Zn, Cd, Ni, and Cu in a single extraction as well as the highest rates of extraction among the studied extractants. The transfer of heavy metals from soil to organic acid solutions is slower than to DTPA and Mehlich-I extractants.Poucos trabalhos têm sido realizados sobre a comparação da taxa de dessorção e da quantidade de metais extraídos sucessivamente por misturas de ácidos orgânicos que imitam a composição da rizosfera e por extratores usados na rotina em amostras de solo tratado com lodo de esgoto. Extrações de Zn, Cd, Ni, Cr e Cu foram realizadas em amostras de um solo de carga variável tratado com lodo de esgoto usando-se sete extratores: quatro misturas com ácidos orgânicos e três extratores usados na rotina (DTPA, Mehlich-I e acetato de amônio). Os resultados para uma única extração, em que o conteúdo de metais extraíveis foi determinado simplesmente extraindo-os do solo uma única vez, bem como após 15 extrações sucessivas, em que o resíduo sólido da primeira extração foi extraído sucessivamente por mais 14 vezes, foram analisados. A extractabilidade dos metais em uma extração simples foi, em geral, como segue: Mehlich-I >; DTPA >; ácidos orgânicos >; NH4OAc. As mais elevadas taxas de extração seguiram a ordem: DTPA >; Mehlich-I >; ácidos orgânicos >; NH4OAc. A maior extração de metais foi obtida com Mehlich-I, enquanto, o DTPA também extraiu alta quantidade de Zn, Cd, Ni e Cu em uma extração simples, bem como obteve as mais altas taxas de extração entre os extratores estudados. A transferência de metais do solo para as soluções de ácidos orgânicos foi mais lenta que para os extratores DTPA e Mehlich-I

Management of the cervical esophagogastric anastomotic stricture

Esophagogastric anastomotic stricture following esophagectomy with a gastric esophageal substitute can be a vexing problem for the patient and treating physician. We describe the clinical practice at a single center with extensive experience in esophageal surgery for management of this complication.http://deepblue.lib.umich.edu/bitstream/2027.42/57525/1/Anastomotic Stricture 2007.pd

Transhiatal esophagectomy in the profoundly obese: implications and experience.

BACKGROUND: Historically, obesity contraindicated an abdominal approach to the esophagogastric junction. The technique of transhiatal esophagectomy (THE) evolved without specific regard to body habitus. The dramatic increase in obese patients requiring an esophagectomy for complications of reflux disease prompted this evaluation of the impact of obesity on the outcomes of esophagectomy to determine whether profound obesity should contraindicate the transhiatal approach. METHODS: We used our Esophagectomy Database to identify 133 profoundly obese patients (body mass index [BMI] > or = 35 kg/m2) from among 2176 undergoing a THE from 1977 to 2006. This group was matched to a randomly selected, non-obese (BMI, 18.5 to 30 kg/m2) control population of 133 patients. Intraoperative, postoperative, and long-term follow-up results were compared retrospectively. RESULTS: Profoundly obese patients had significantly greater intraoperative blood loss (mean, 492.2 mL versus 361.8 mL, p = 0.001), need for partial sternotomy (18 versus 3, p = 0.001), and frequency of recurrent laryngeal nerve injury (6 versus 0, p = 0.04). The two groups did not differ significantly in the occurrence of chylothorax, wound infection, or dehiscence rate; length of hospital stay or need for intensive care unit stay; or hospital or operative mortality. Follow-up results for dysphagia, dumping, regurgitation, and overall functional score were also comparable between the two groups. CONCLUSIONS: With appropriate instrumentation, transhiatal esophagectomy in obese patients has similar morbidity and outcomes as in non-obese patients. Obesity, even when profound, does not contraindicate a transhiatal esophagectomy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57503/6/Scipione 2007.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/57503/5/Benign BMI Control.txthttp://deepblue.lib.umich.edu/bitstream/2027.42/57503/4/Benign BMI CS07.txthttp://deepblue.lib.umich.edu/bitstream/2027.42/57503/3/CA BMI Control no pt id.txthttp://deepblue.lib.umich.edu/bitstream/2027.42/57503/2/CA BMI 35 CS.tx

Center-surround vs. distance-independent lateral connectivity in the olfactory bulb

Lateral neuronal interactions are known to play important roles in sensory information processing. A center-on surround-off local circuit arrangement has been shown to play a role in mediating contrast enhancement in the visual, auditory, and somatosensory systems. The lateral connectivity and the influence of those connections have been less clear for the olfactory system. A critical question is whether the synaptic connections between the primary projection neurons, mitral and tufted (M/T) cells, and their main inhibitory interneurons, the granule cells (GCs), can support a center-surround motif. Here, we study this question by injecting a “center” in the glomerular layer of the olfactory bulb (OB) with a marker of synaptic connectivity, the pseudorabies virus (PRV), then examines the distribution of labeling in the “surround” of GCs. We use a novel method to score the degree to which the data fits a center-surround model vs. distance-independent connectivity. Data from 22 injections show that M/T cells generally form lateral connections with GCs in patterns that lie between the two extremes