Partial Reconstruction of the Ergot Alkaloid Pathway by Heterologous Gene Expression in Aspergillus nidulans

Ergot alkaloids are pharmaceutically and agriculturally important secondary metabolites produced by several species of fungi. Ergot alkaloid pathways vary among different fungal lineages, but the pathway intermediate chanoclavine-I is evolutionarily conserved among ergot alkaloid producers. At least four genes, dmaW, easF, easE, and easC, are necessary for pathway steps prior to chanoclavine-I; however, the sufficiency of these genes for chanoclavine-I synthesis has not been established. A fragment of genomic DNA containing dmaW, easF, easE, and easC was amplified from the human-pathogenic, ergot alkaloid-producing fungus Aspergillus fumigatus and transformed into Aspergillus nidulans, a model fungus that does not contain any of the ergot alkaloid synthesis genes. HPLC and LC-MS analyses demonstrated that transformed A. nidulans strains produced chanoclavine-I and an earlier pathway intermediate. Aspergillus nidulans transformants containing dmaW, easF, and either easE or easC did not produce chanoclavine-I but did produce an early pathway intermediate and, in the case of the easC transformant, an additional ergot alkaloid-like compound. We conclude that dmaW, easF, easE, and easC are sufficient for the synthesis of chanoclavine-I in A. nidulans and expressing ergot alkaloid pathway genes in A. nidulans provides a novel approach to understanding the early steps in ergot alkaloid synthesis

Substrate-Dependent Photoconductivity Dynamics in a High-Efficiency Hybrid Perovskite Alloy

Films of (FA0.79MA0.16Cs0.05)0.97Pb(I0.84Br0.16)2.97 were grown over TiO2, SnO2, indium tin oxide (ITO), and NiO. Film conductivity was interrogated by measuring the in-phase and out-of-phase forces acting between the film and a charged microcantilever. We followed the films’ conductivity versus time, frequency, light intensity, and temperature (233−312 K). Perovskite conductivity was high and light-independent over ITO and NiO. Over TiO2 and SnO2, the conductivity was low in the dark,increased with light intensity, and persisted for 10’s of seconds after the light was removed. At an elevated temperature over TiO2, the rate of conductivity recovery in the dark showed an activated temperature dependence (Ea= 0.58eV). Surprisingly, the light-induced conductivity over TiO2 and SnO2 relaxed essentially instantaneously at a low temperature. We use a transmission-line model for mixed ionic−electronic conductors to show that the measurements presented are sensitive to the sum of electronic and ionic conductivities. We rationalize the seemingly incongruous observations using the idea that holes, introduced either by equilibration with the substrate or via optical irradiation, create iodide vacancies

Substrate-dependent Photoconductivity Dynamics in a High-efficiency Hybrid Perovskite Alloy

Films of (FA$_{0.79}$MA$_{0.16}$Cs$_{0.05}$)$_{0.97}$Pb(I$_{0.84}$Br$_{0.16}$)$_{2.97}$ were grown over TiO$_{2}$, SnO$_{2}$, ITO, and NiO. Film conductivity was interrogated by measuring the in-phase and out-of-phase forces acting between the film and a charged microcantilever. We followed the films' conductivity vs. time, frequency, light intensity, and temperature (233 to 312 K). Perovskite conductivity was high and light-independent over ITO and NiO. Over TiO$_{2}$ and SnO$_{2}$, the conductivity was low in the dark, increased with light intensity, and persisted for 10's of seconds after the light was removed. At elevated temperature over TiO$_{2}$, the rate of conductivity recovery in the dark showed an activated temperature dependence (E$_{a}$ = 0.58 eV). Surprisingly, the light-induced conductivity over TiO$_{2}$ and SnO$_{2}$ relaxed essentially instantaneously at low temperature. We use a transmission-line model for mixed ionic-electronic conductors to show that the measurements presented are sensitive to the sum of electronic and ionic conductivities. We rationalize the seemingly incongruous observations using the idea that holes, introduced either by equilibration with the substrate or via optical irradiation, create iodide vacancies

External Tank (ET) Foam Thermal/Structural Analysis Project

An independent study was performed to assess the pre-launch thermally induced stresses in the Space Shuttle External Tank Bipod closeout and Ice/Frost ramps (IFRs). Finite element models with various levels of detail were built that included the three types of foam (BX-265, NCFI 24-124, and PDL 1034) and the underlying structure and bracketry. Temperature profiles generated by the thermal analyses were input to the structural models to calculate the stress levels. An area of high stress in the Bipod closeout was found along the aluminum tank wall near the phenolic insulator and along the phenolic insulator itself. This area of high stress might be prone to cracking and possible delamination. There is a small region of slightly increased stress in the NCFI 24-124 foam near its joint with the Bipod closeout BX-265 foam. The calculated stresses in the NCFI 24-124 acreage foam are highest at the NCFI 24-124/PDL 1034/tank wall interface under the LO2 and LH2 IFRs. The highest calculated stresses in the LH2 NCFI 24-124 foam are higher than in similar locations in the LO2 IFR. This finding is consistent with the dissection results of IFRs on ET-120

Robotic-arm assisted lateral unicompartmental knee arthroplasty: 5-Year outcomes & survivorship

INTRODUCTION: Robotic-arm assisted unicompartmental knee arthroplasty (RA-UKA) has demonstrated accurate component positioning and excellent outcomes for medial components. However, there is a paucity of literature on lateral compartment RA-UKA. The purpose of our study was to assess the midterm clinical outcomes and survivorship of lateral RA-UKA. METHODS: This study was a retrospective review of a single-center prospectively maintained cohort of 33 patients (36 knees) indicated for lateral UKA. Perioperative, and postoperative two- and five-year Knee injury Osteoarthritis Outcome Score (KOOS), Western Ontario and McMaster Universities Osteoarthritis Score (WOMAC), and Forgotten Joint Score (FJS) patient reported outcome measures were collected. Five-year follow-up was recorded in 29 patients (32 knees). RESULTS: Mean follow up was 5.1 ± 0.1 years. Mean age and BMI was 70.9 ± 7.2 years and 29.0 ± 4.2 kg/m2, respectively. At discharge, mean distance walked was 273.4 ± 70.4 feet, and mean pain score was 2.0 ± 2.5. At 2-year follow up, mean KOOS, WOMAC, and FJS were 75.1 ± 13.5, 15.0 ± 7.2, and 81.0 ± 23.3, respectively. At 5-year follow up, mean KOOS, WOMAC, and FJS were 75.3 ± 14.6, 14.9 ± 5.0, and 75.8 ± 27.4, respectively. Mean change in KOOS and WOMAC were 35.6 ± 27.1 and 11.7 ± 13.4 (p\u3c .001 and p\u3c .001). 94% of patients were very satisfied/satisfied, 3% neutral, and 3% dissatisfied. 91% met activity expectations, and 59% were more active than before. Survivorship was 100% at 5 years. DISCUSSION: In this study, lateral RA-UKA demonstrated significantly improved clinical outcomes, high patient satisfaction, met expectations, and excellent functional recovery at midterm follow up. Comparative studies are needed to determine differences between robotic-assisted and conventional lateral UKA, as well as TKA

5-Year Survivorship and Outcomes of Robotic-Arm-Assisted Medial Unicompartmental Knee Arthroplasty

Purpose: While unicompartmental knee arthroplasty (UKA) has demonstrated benefits over total knee arthroplasty (TKA) in selected populations, component placement continues to be challenging with conventional surgical instruments, resulting in higher early failure rates. Robotic-arm-assisted UKA (RA-UKA) has shown to be successful in component positioning through preop planning and intraop adjustability. The purpose of this study is to assess the 5-year clinical outcomes of medial RA-UKA. Methods: This study was a retrospective review of a single-center prospectively maintained cohort of 133 patients (146 knees) indicated for medial UKA from 2009 to 2013. Perioperative data and 2- and 5-year Knee injury Osteoarthritis Outcome Score (KOOS), Western Ontario and McMaster Universities Osteoarthritis Score (WOMAC), and Forgotten Joint Score (FJS) outcome measures were collected. Five-year follow-up was recorded in 119 patients (131 knees). Results: Mean follow-up was 5.1 ± 0.2 years. Mean age and BMI were 68.0 ± 8.1 years and 29.3 ± 4.7 kg/m(2), respectively. At 2-year follow-up, mean KOOS, WOMAC, and FJS were 71.5 ± 15.3, 14.3 ± 7.9, and 79.1 ± 25.8, respectively. At 5-year follow-up, mean KOOS, WOMAC, and FJS were 71.6 ± 15.2, 14.2 ± 7.9, and 80.9 ± 25.1, respectively. Mean change in KOOS and WOMAC was 34.6 ± 21.4 and 11.0 ± 13.6, respectively (p \u3c 0.001 and p \u3c 0.001). For patient satisfaction at last follow-up, 89% of patients were very satisfied/satisfied and 5% were dissatisfied. For patient activity expectations at last follow-up, 85% met activity expectations, 52% were more active than before, 25% have the same level of activity, 23% were less active than before, and 89% were walking without support. All patients returned to driving after surgery at a mean 15.2 ± 9.4 days. Survivorship was 95% (95% CI 0.91-0.98) at 5 years. One knee (1%) had a patellofemoral revision, two knees (1.3%) were revised to different partial knee replacements, and five knees (3.4%) were converted to TKA. Conclusion: Overall, medial RA-UKA demonstrated improved patient-recorded outcomes, high patient satisfaction, met expectations, and excellent functional recovery. Midterm survivorship was excellent. Longitudinal follow-up is needed to evaluate long-term outcomes of robotic-arm-assisted UKA procedures

Flow Cytometric Analysis of Hematopoietic Populations in Rat Bone Marrow. Impact of Trauma and Hemorrhagic Shock

Severe injury and hemorrhagic shock (HS) result in multiple changes to hematopoietic differentiation, which contribute to the development of immunosuppression and multiple organ failure (MOF). Understanding the changes that take place during the acute injury phase may help predict which patients will develop MOF and provide potential targets for therapy. Obtaining bone marrow from humans during the acute injury phase is difficult so published data is largely derived from peripheral blood samples, which infer bone marrow changes that reflect the sustained inflammatory response. This preliminary and opportunistic study investigated leucopoietic changes in rat bone marrow 6 hours following traumatic injury and HS. Terminally anesthetized male Porton Wistar rats were allocated randomly to receive a sham operation (cannulation with no injury) or femoral fracture and HS. Bone marrow cells were flushed from rat femurs and immunophenotypically stained with specific antibody panels for lymphoid (CD45R, CD127, CD90, IgM) or myeloid (CD11b, CD45, RP-1) lineages. Subsequently, cell populations were fluorescence activated cell sorted for morphological assessment. Stage-specific cell populations were identified using a limited number of antibodies and leucopoietic changes were determined 6 hours following trauma and HS. Myeloid sub-populations could be identified by varying levels CD11b expression, CD45 and RP-1. Trauma and HS resulted in a significant reduction in total CD11b+ myeloid cells including both immature (RP-1(-)) and mature (RP-1+) granulocytes. Multiple B-cell lymphoid subsets were identified. The total % of CD90+ subsets remained unchanged following trauma and HS, but there was a reduction in the numbers of maturing CD90(-) cells suggesting movement into the periphery

A parametric integer programming algorithm for bilevel mixed integer programs

We consider discrete bilevel optimization problems where the follower solves an integer program with a fixed number of variables. Using recent results in parametric integer programming, we present polynomial time algorithms for pure and mixed integer bilevel problems. For the mixed integer case where the leader's variables are continuous, our algorithm also detects whether the infimum cost fails to be attained, a difficulty that has been identified but not directly addressed in the literature. In this case it yields a ``better than fully polynomial time'' approximation scheme with running time polynomial in the logarithm of the relative precision. For the pure integer case where the leader's variables are integer, and hence optimal solutions are guaranteed to exist, we present two algorithms which run in polynomial time when the total number of variables is fixed.Comment: 11 page

A "Politically Robust" Experimental Design for Public Policy Evaluation, with Application to the Mexican Universal Health Insurance Program

We develop an approach to conducting large scale randomized public policy experiments intended to be more robust to the political interventions that have ruined some or all parts of many similar previous efforts. Our proposed design is insulated from selection bias in some circumstances even if we lose observations; our inferences can still be unbiased even if politics disrupts any two of the three steps in our analytical procedures; and other empirical checks are available to validate the overall design. We illustrate with a design and empirical validation of an evaluation of the Mexican Seguro Popular de Salud (Universal Health Insurance) program we are conducting. Seguro Popular, which is intended to grow to provide medical care, drugs, preventative services, and financial health protection to the 50 million Mexicans without health insurance, is one of the largest health reforms of any country in the last two decades. The evaluation is also large scale, constituting one of the largest policy experiments to date and what may be the largest randomized health policy experiment ever.Governmen

Subnanogram proteomics: impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ∼3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ∼ 95% for 0.5 ng samples and by ∼42% for 2 ng samples. Using the best combination of the above variables, we were able to identify >3,000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. The present ultrasensitive LC-MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells