Derivation of human trophoblast stem cells from placentas at birth.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3ß) inhibitor CHIR99021, the transforming growth factor-beta (TGFß) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology.

Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFß) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFß signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFß inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFß inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro.

National Cancer Institute (NCI) Program for Natural Products Discovery: Rapid Isolation and Identification of Biologically Active Natural Products from the NCI Prefractionated Library.

An automated, high-capacity, and high-throughput procedure for the rapid isolation and identification of biologically active natural products from a prefractionated library is presented. The semipreparative HPLC method uses 1 mg of the primary hit fraction and produces 22 subfractions in an assay-ready format. Following screening, all active fractions are analyzed by NMR, LCMS, and FTIR, and the active principle structural classes are elucidated. In the proof-of-concept study, we show the processes involved in generating the subfractions, the throughput of the structural elucidation work, as well as the ability to rapidly isolate and identify new and biologically active natural products. Overall, the rapid second-stage purification conserves extract mass, requires much less chemist time, and introduces knowledge of structure early in the isolation workflow.

The NCI library of traditional Chinese medicinal plant extracts - Preliminary assessment of the NCI-60 activity and chemical profiling of selected species.

Botanical-based natural products are an important resource for medicinal drug discovery and continue to provide diverse pharmacophores with therapeutic potential against cancer and other human diseases. A prototype Traditional Chinese Medicine (TCM) plant extract library has been established at the US National Cancer Institute, which contains both the organic and aqueous extracts of 132 authenticated medicinal plant species that collectively represent the potential therapeutic contents of most commonly used TCM herbal prescriptions. This library is publicly available in 96- and 384- well plates for high throughput screening across a broad array of biological targets, as well as in larger quantities for isolation of active chemical ingredients. Herein, we present the methodology used to generate the library and the preliminary assessment of the anti-proliferative activity of this crude extract library in NCI-60 human cancer cell lines screen. Particularly, we report the chemical profiling and metabolome comparison analysis of four commonly used TCM plants, namely Brucea javanica, Dioscorea nipponica, Cynanchum atratum, and Salvia miltiorrhiza. Bioassay-guided isolation resulted in the identification of the active compounds, and different extraction methods were compared for their abilities to extract cytotoxic compounds and to concentrate biologically active natural products.

NCI Program for Natural Product Discovery: A Publicly-Accessible Library of Natural Product Fractions for High-Throughput Screening.

The US National Cancer Institute's (NCI) Natural Product Repository is one of the world's largest, most diverse collections of natural products containing over 230,000 unique extracts derived from plant, marine, and microbial organisms that have been collected from biodiverse regions throughout the world. Importantly, this national resource is available to the research community for the screening of extracts and the isolation of bioactive natural products. However, despite the success of natural products in drug discovery, compatibility issues that make extracts challenging for liquid handling systems, extended timelines that complicate natural product-based drug discovery efforts and the presence of pan-assay interfering compounds have reduced enthusiasm for the high-throughput screening (HTS) of crude natural product extract libraries in targeted assay systems. To address these limitations, the NCI Program for Natural Product Discovery (NPNPD), a newly launched, national program to advance natural product discovery technologies and facilitate the discovery of structurally defined, validated lead molecules ready for translation will create a prefractionated library from over 125,000 natural product extracts with the aim of producing a publicly-accessible, HTS-amenable library of >1,000,000 fractions. This library, representing perhaps the largest accumulation of natural-product based fractions in the world, will be made available free of charge in 384-well plates for screening against all disease states in an effort to reinvigorate natural product-based drug discovery.

therapy services: from ancillary to necessary.

Physical and occupational therapy and speech language pathology services are assuming an increasingly important role as healthcare providers transition to value-based care. Finance leaders can support the efforts of therapy leaders by working with them to: Determine priorities regarding level of care. Establish appropriate staffing and productivity metrics. Promote accurate and thorough charge-capture efforts. Broaden the referral base for therapy services

From the front line to the bottom line: building revenue integrity.

To improve trends in accounts receivable and a hospital's bottom line without fear of penalty or repayment, organizations should expand the definition of the revenue cycle team by: Engaging front-line clinical and business personnel. Training personnel to understand the roles they play in revenue integrity. Creating scorecards with measurable goals to promote accountability. Monitoring the outcomes and defining real-time, actionable responses to negative variances.

Feasibility of voluntary menu labeling among locally owned restaurants.

In 2007, Tacoma-Pierce County Health Department launched a restaurant menu labeling project called SmartMenu. The objective was to recruit locally owned restaurants to voluntarily post basic nutrition information on their menus or menu boards. Participating restaurants submitted recipes to an independent contractor for nutritional analysis and agreed to post calorie, fat, carbohydrate, and sodium values on new menus within 90 days of receiving results. Vigorous recruitment efforts by the Health Department between June 2007 and September 2008 included free advertising, consultation with a Registered Dietitian, and free nutritional analysis. By the end of 2008, a total of 24 restaurants participated in the program. Significant barriers to participation included infrequent use of standardized recipes, perceived business risk of labeling, and low perceived customer demand for nutrition information. Key program elements, recruitment strategies, and costs are discussed. Results have important implications for future efforts to increase the adoption of menu labeling by locally owned and operated restaurants.

Overcoming problems of compound storage in DMSO: solvent and process alternatives.

The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration.

The use of assessment in promoting secondhand smoke policy in a local health jurisdiction.

BACKGROUND: Clean indoor air regulations reduce both exposure to secondhand smoke and use of tobacco, two significant causes of death in the United States. In 2003, the Board of Health in Pierce County, Washington State, adopted a resolution prohibiting indoor smoking in all public places. Assessment activities were used in three key steps during the secondhand smoke policy development process: (1) setting prevention priorities, (2) monitoring and evaluating interventions, and (3) adopting local policy change. METHODS: Step 1 included calculating attributable risks for morbidity and mortality caused by preventable health behaviors. Step 2 involved designing logic models and outcomes-based evaluations to collect and analyze data from prevention efforts. Surveillance of restaurants documented voluntary adoption of smoke-free policies. Step 3 included conducting telephone surveys to track public support for tobacco policy approaches. FINDINGS: Results demonstrated tobacco's high impact on morbidity and mortality, illustrated a plateau of restaurants' voluntary smoke-free policies, and identified growing public support for secondhand smoke policy. Assessment results were included in multiple policy and support documents and cited by Board of Health members during policy adoption. CONCLUSIONS: Assessment data contributed critical support to local public health decision makers during key steps of a lengthy secondhand smoke policy development process.

The rehabilitation of rehab.

To meet the needs of rehabilitation inpatients while complying with changes in regulatory requirements, providers may need to: Revisit discharge planning, Ensure high-quality documentation, Have a centralized post-acute intake process, Assess the current continuum, Be active in educating referral sources.

A heart for change.

The effectiveness of change management is driven by six factors: A clear and consistent message from the top. A palpable connection between leaders and clinicians. Departments that perform their functions well, but also work in sync with other departments. A healthy level of resistance in the change process. Effective communication. A model for monitoring the change process and responding early to variances.

Minding your business.

There are a number of ways that hospitals can enhance financial perfomance from their outpatient physical therapy services: Provide training for therapists on Medicare's "eight minute rule". Differentiate between service-based and time-based CPT codes on the charge ticket. Have a professional review CPT documentation before charges are submitted. Implement a program to prevent and manage no-shows and cancellations. Match staffing level with productivity. Pay careful attention to detail when documenting plans of care and preparing daily treatment notes.

Evaluation of a community-based handgun safe-storage campaign.

OBJECTIVE: Safe storage of firearms has been recommended as a means of preventing gun-related pediatric injuries, yet few interventions have led to significant improvements in storage practices. This study examined a multifaceted community education campaign to promote safe handgun storage and the campaign's impact on firearm locking and loading practices in households with children. METHODS: Beginning in 1997, a safe-storage campaign consisting of television and radio announcements, educational materials, billboards, and discount coupons for lock boxes was conducted in King County, Washington. The campaign evaluation used a quasi-experimental design and compared the intervention site with 9 control counties outside Washington State and west of the Mississippi River. Cross-sectional, random-digit-dial telephone surveys of handgun-owning households with children were conducted in all study counties both before the intervention in 1996 (n = 302) and again in 2001 (n = 255). The main analyses assessed whether greater improvements in household firearm-storage practices occurred between 1996 and 2001 in the intervention, compared with the control, counties. Primary outcomes were based on up to 3 handguns per household and included (1) all stored with trigger locks, lock boxes, or gun safes (formal locking devices), (2) all stored in lock boxes or gun safes, (3) any stored loaded, (4) any stored loaded without a formal locking device, and (5) any stored loaded and not in a lock box or gun safe. Data were also collected on up to 1 long gun per household; long-gun outcomes included (1) stored with a trigger lock or gun safe and (2) stored loaded. RESULTS: Overall, handguns and long guns were generally more likely to be stored locked and less likely to be loaded in 2001 compared with 1996, with these trends seeming to be more consistent in the intervention county. Even so, more than one quarter of households with children and handguns in 2001 failed to store all of their handguns with a formal locking device, and up to 8% continued to possess at least 1 loaded handgun that was not stored with a formal device. The majority of households that stored their handguns with formal devices used lock boxes or gun safes. Storage of handguns in lock boxes or gun safes became more common in both the intervention (adjusted odds ratio [aOR]: 1.71; 95% confidence interval [CI]: 1.03-2.84) and control households (aOR: 1.66; 95% CI: 1.01-2.72) between 1996 and 2001. None of the other measured changes reached statistical significance, such as storing any household handgun loaded (aOR: 0.71; 95% CI: 0.35-1.42 [intervention]; aOR: 1.08; 95% CI: 0.58-2.00 [control]) or keeping any household handgun loaded and not stored in a lock box or gun safe (aOR: 0.59; 95% CI: 0.22-1.55 [intervention]; aOR: 0.67; 95% CI: 0.30-1.49 [control]). Moreover, the intervention county did not experience significantly greater overall improvements in household storage practices for handguns or long guns than did control counties. CONCLUSIONS: In both the intervention and control counties, households were more likely to lock all handguns in 2001 compared with 1996. After accounting for temporal trends, this educational campaign, combined with economic incentives to purchase lock boxes, did not seem to significantly change safe storage practices in households with handguns and children. Even if the campaign did result in small improvements in firearm safe storage, simultaneous national and state-specific gun-safety activities or legislative efforts may have drawn increasing attention to gun-related issues in the control counties, thereby making it more difficult to identify effects of our specific handgun storage intervention.