In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. Here we use conjugation, a natural DNA horizontal transfer process among bacterial species, to deliver an engineered CRISPR interference (CRISPRi) system for targeting specific genes in recipient Escherichia coli cells. We show that delivery of the CRISPRi system is successful and can specifically repress a reporter gene in recipient cells, thereby establishing a new tool for gene regulation across bacterial cells and potentially for bacterial population control.
Image guided radiation therapy (IGRT) using bony anatomy for bladder cancer requires the use of large population-based planning target volume (PTV) margins to compensate for geometric uncertainties. This may result in a large volume of normal tissue being irradiated unnecessarily. Identification of the clinical target volume (CTV) is also a challenge during target delineation and treatment position verification. This study describes the use of lipiodol (Guerbet, US) and cone beam computed tomography (CBCT) in deriving patient-specific PTV (PS-PTV) for partial bladder IGRT.
Differentiating heart failure (HF) induced renal dysfunction (RD) from intrinsic kidney disease is challenging. It has been demonstrated that biomarkers such as B-type natriuretic peptide (BNP) or the blood urea nitrogen to creatinine ratio (BUN/Creat) can identify high vs. low risk RD. Our objective was to determine if combination these biomarkers could further improve risk stratification and clinical phenotyping of patients with RD and HF.
Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. An alternative non-contact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of calibration curve and QCs in bioanalysis.
Alisertib (MLN8237) is an investigational potent Aurora A kinase inhibitor currently under clinical trials for hematological and nonhematological malignancies. Nonclinical investigation showed that alisertib is a highly permeable compound with high plasma protein binding, low plasma clearance, and moderate volume of distribution in rats, dogs, monkeys and chimpanzees. Consistent with the above properties, the oral bioavailability in animals was greater than 82%. The predicted human oral pharmacokinetic (PK) profile was constructed using allometric scaling of plasma clearance and volume of distribution in the terminal phase from animals. The chimpanzee PK profiles were extremely useful to model absorption rate constant, which was assumed to be similar to that in humans, based on the fact that chimpanzees are phylogenetically closest to humans. The human plasma clearance was projected to be low of 0.12 L/hr/kg, with half-life of approximately 10 hr. For human efficacious dose estimation, the tumor growth inhibition as a measure of efficacy (E) was assessed in HCT116 xenograft mice at several oral QD or BID dose levels. Additionally, subcutaneous mini-pump infusion studies were conducted to assess mitotic index in tumor samples as a pharmacodynamic (PD) marker. PK/PD/E modeling showed that for optimal efficacy and PD in the xenograft mice maintaining a plasma concentration exceeding 1 µM for at least 8-12 hr would be required. These values in conjunction with the projected human PK profile estimated the optimal oral dose of approximately 103 mg QD or 62.4 mg BID in humans. Notably, the recommended Phase 2 dose being pursued in the clinic is close to the projected BID dose.
Column-related carryover affects both accuracy and precision in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In this work, a novel straightforward dynamic flush method to reduce the column-related carryover was developed by alternating the column flow direction of liquid chromatographic separation with a Valco switching valve and pristine instrument control software. By alternating the column flow direction, a fresh inlet is always in line to accommodate sample injection and stacking. In addition, the contaminated column inlet from the previous run is switched to the outlet position for a flush with a gradient during the next sample run. In this way, the column-related carryover can be reduced effectively without additional blank runs. It also minimizes the carryover risk between the adjacent unknown samples. The column-related carryover of the tested "sticky" compounds was reduced by 52.3-94.4% compared with the non-dynamic flush method under the same experimental conditions. The performance and reproducibility of high-performance liquid chromatography (HPLC) separation in terms of the retention time shift and peak shape are not compromised under the dynamic flush even after over 300 consecutive injections. The described novel method is simple and easy to implement for compounds with column-related carryover.
Neck pain is an extremely common symptom with many possible etiologies. A substantial number of patients are turning to complementary and alternative medicine (CAM). Low-quality evidence supports the beneficial effects of CAM. Feldenkrais, massage therapy, and spinal manipulation are discussed in detail. Complications are generally benign and self-limited, although occasional catastrophic consequences have been documented. Despite the favorable opinion many rheumatologists have of some CAM therapy, many patients are not disclosing CAM use to their medical providers. By expressing interest, asking questions, and taking a shared-decision-making approach, providers can encourage disclosure and provide valuable input.
Using various physicochemical methods of analysis, we examined human hair in its virgin and delipidized state. Free lipids were removed by a solvent extraction technique (covalently bound lipids were not removed) using a series of solvents with varying polarity. We analyzed the surface properties of hair by conducting mechanical combing and dynamic contact angle analysis. In addition, we used inverse gas chromatography surface energy analysis to explore the chemical composition of the hair surface based on interactions of various nonpolar and polar probes with biological molecules residing on the hair surface. Further, we investigated the importance that free lipids play in the internal structural properties of hair using dynamic scanning calorimetry and tensile strength measurements. The microstructure of the hair surface was probed by atomic force microscopy, whereas the lipid content of hairs morphological components was determined by infrared spectroscopic imaging. We also monitored the water management properties of virgin and delipidized hair by dynamic vapor sorption, which yielded unique water sorption isotherms for each hair type. Using all these techniques, differences were found in the chemical composition and physical behavior of virgin and delipidized hair. To better understand the influence of hair lipid composition on hair styling treatments, we conducted mechanical analyses of hair shaped into omega loops to determine the stiffness, elasticity, and flexibility of hair-polymer assemblies. Although there were no discernible differences between untreated virgin and delipidized hair, in terms of stiffness and elasticity, we found that treatment with hair styling agents produced different effects depending on the hair type used. Likewise, streaming potential measurements were carried out to monitor the binding capacity of rinse-off treatments on virgin and delipidized hair. Using this technique, we monitored the surface potential of hair and found significant differences in the binding behavior of cationic polymers and surfactants (polyquaternium-55 and quaternium-26) on both hair types.
Alisertib (MLN8237) is an investigational inhibitor of Aurora A kinase (AAK). Aurora A plays an essential role in the regulation of spindle assembly and chromosome alignment during mitosis. Inhibition of Aurora A by alisertib in tissue culture has previously been demonstrated to lead to improper chromosomal alignment and disruption of spindle organization, resulting in a transient mitotic delay. The spindle organization defects induced by alisertib have been used to develop a pharmacodynamic (PD) assay for Aurora A inhibition based on the percentage of mitotic cells with proper chromosomal alignment at the metaphase plate (% aligned spindles, abbreviated as AS). The transient mitotic delay that occurs with AAK inhibition permits the use of the mitotic index (the fraction of cells in the population currently undergoing mitosis, abbreviated as MI) as an additional PD assay. When the two PD assays were used in Phase I clinical trials, the reduction in AS was strongly correlated with dose levels and exposures in patients from single time point PD measurements; however, MI failed to show any correlation. To further understand this clinical finding, we constructed PK/PD/efficacy models for AS and MI that can precisely capture the temporal dynamics of the PD markers from in vivo xenograft studies.
The choice of environmental conditions when conducting antiperspirant studies greatly affects the quantity of sweat output. Our initial goal in this work was to develop an in-house procedure to test the efficacy of antiperspirant products using replica techniques in combination with image analysis. To ameliorate the skin replica method, we conducted rheological studies using dynamic mechanical analysis of the replica formulation. In terms of sweat output quantification, our preliminary results revealed a considerable amount of variation using the replica technique, leading us to conduct more fundamental studies of the factors that influence sweating behavior and how to best design the experimental strategy. In accordance with the FDAs protocol for antiperspirant testing, we carried out gravimetric analyses of axillae sweating under a variety of environmental conditions including temperature and humidity control. Subjects were first acclimatized in an environmentally controlled room for 30 min, and then placed in a sauna for an additional 30 or 45 min, depending on which test we administered. In Test 1 (30 min total in the sauna), the first 10 min in the sauna was another equilibration period, followed by a 20 min sweat production stage. We monitored axillae sweating during the last 20 min in the sauna by gravimetric analysis. At time (t) = 30 min in the sauna, skin replicas were taken and later analyzed using imaging and image analysis techniques. Test 1 was carried out on over 25 subjects, both male and female, from various racial backgrounds. In Test 2, subjects spent 45 min in the sauna after the initial 30-min period in the environmental room. During the 45 min, we obtained gravimetric readings of absorbent pads placed in the axillae. We conducted studies at various temperature and relative humidity settings. We also studied the influence of several external parameters on sudoriferous activity. Test 2 was a range-finding experiment on two subjects to determine the optimized environmental conditions for the hot room procedure. In addition to the replica and gravimetric techniques, we also measured flux density to determine the onset of firing of sweat glands to ensure that our environmental preconditioning step (30 min in the environmental room) brought subjects to the point that their sweat glands were activated. Although flux density measurements are usually carried out to determine transepidermal water loss (TEWL), we found that they can be equally useful for monitoring the onset of sweat production. Thermal infrared imaging experiments were also carried out allowing us to generate full-body images of subjects containing anatomical thermal distribution data with high accuracy. Overall, we conclude that our in-house hot room procedure offers much potential as an effective and cost-efficient screening tool for narrowing copious antiperspirant formulations to a select few for expensive clinical evaluation.
The ability to engineer novel functionality within cells, to quantitatively control cellular circuits, and to manipulate the behaviors of populations, has many important applications in biotechnology and biomedicine. These applications are only beginning to be explored. In this review, we advocate the use of feedback control as an essential strategy for the engineering of robust homeostatic control of biological circuits and cellular populations. We also describe recent works where feedback control, implemented in silico or with biological components, was successfully employed for this purpose.
To examine associations between maternal employment and time spent engaging in nutrition-related behaviours among mothers and children using a nationally representative sample of households in West and East Germany.
Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.
Of the multitude of treatment options for the management of neck pain, no obvious single treatment modality has been shown to be most efficacious. As such, the clinician should consider alternative treatment modalities if a modality is engaging, available, financially feasible, potentially efficacious, and is low risk for the patient. As evidence-based medicine for neck pain develops, the clinician is faced with the challenge of which treatments to encourage patients to pursue. Treatment modalities explored in this article, including chiropractic, acupuncture, TENS, massage, yoga, Tai Chi, and Feldenkrais, represent reasonable complementary and alternative medicine methods for patients with neck pain.
The purpose of this study is to assimilate evidence regarding quality of care received at nurse-managed clinics (NMCs), particularly a pediatric NMC that provides health care for the underserved pediatric population.
Hair is frequently exposed to environmental stresses and chemical insults that result in damage to its internal structure and its outer cuticular components. Spectrofluorescence is a useful tool to monitor the health of biological tissues as it can measure the level of tryptophan (Trp), which is representative of protein integrity. In addition to Trp fluorescence, several other fluorophores are also present in hair and are believed to be attributed to kynurenenine, N-formylkynurenine, and 3-hydroxykynurenine, which are known metabolic and degradation products of Trp that are affected by environmental stresses normally experienced by hair. In this work, we were able to construct an endogenous fingerprint of fluorescent compounds present in hair by employing a range of excitation wavelengths from 270 nm to 450 nm with a resolution of 2 nm. As a result, we generated surface plots of fluorescence emission as a function of excitation and emission wavelengths (excitation-emission matrices). Thus, we were able to profile the levels of various structural molecules in hair before and after exposure to UV irradiation and thermal straightening irons as well as to chemical treatment such as bleaching and straightening.
A pilot study of anti-human leukocyte antigen (HLA)-DR monoclonal antibody (mAb) in dogs with lymphoma was undertaken to verify the suitability of a canine model to address therapeutically relevant endpoints prior to a full trial in dogs, and ultimately human investigation. In vitro studies demonstrated that L243, a murine IgG1 anti-HLA-DR, binds to normal and malignant canine lymphocytes and induces apoptosis in canine lymphoma cells. Moreover, L243 was administered safely to normal dogs and dogs with lymphoma, and bound to malignant cells in nodal tissue. Preliminary evidence of transient disease stabilization was observed in a subset of dogs with advanced-stage lymphoma following L243 immunotherapy. hL243?4P (IMMU-114), a humanized IgG4 anti-HLA-DR, currently under evaluation preclinically for human trials, was also shown to bind malignant canine lymphocytes, and safety and pharmacokinetic data from the administration of IMMU-114 to normal dogs indicate similar behavior to L243 in these assessments. These findings provide a rationale for the use of dogs with lymphoma in safety and efficacy evaluations of anti-HLA-DR mAbs for both veterinary and human applications.
Recent empirical work in the obesity literature has highlighted the role of the built environment and its potential influence in the increasing prevalence of obesity in adults and children. One feature of the built environment that has gained increasing attention is the role of access to chain grocers and their impact on body mass index (BMI). The assessment of the impacts of spatial access to chain grocers on BMI is complicated by two empirical regularities in the data. There is evidence that health outcomes such as BMI are clustered in space and that there is spatial dependence across individuals. In this article, we use an econometric model that takes into account the spatial dependence, and we allow the effect of access to differ for a person depending on whether he or she lives in a low-income community or peer group. We categorize this community using the characteristics of the people who immediately surround the individual rather than using census tracts. Using georeferenced survey data on adults in Marion County, Indiana, we find that the effect of improvements in chain grocer access on BMI varies depending on community characteristics.
The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.
Research on malnutrition typically focuses on extreme cases which pose the greatest individual health risks, but researchers comparing populations might find that variation in mild malnutrition conveys valuable information about public health. This paper constructs and compares new measures of the prevalence, depth and severity of both mild and extreme underweight in children from three months to three years of age, as measured by 130 DHS surveys for 53 countries over a period from 1986 to 2006. We find that variance in mild underweight has a larger and more robust correlation with child mortality than variance in severe underweight, and is itself more closely correlated with local agricultural output, over a wide range of regression specifications. We conclude that the prevalence of mild underweight deserves greater attention as a useful signal of changing public health conditions among preschool children in developing countries.
Several states and local communities have started to experiment with policy initiatives that affect the built-up environment in an attempt to decrease the prevalence of obesity. The focus of these policy measures has generally been to eliminate geographical disparities in access to food. Recent policy proposals include the use of zoning laws to create a healthier food environment by providing incentives for chain grocers to open stores in disadvantaged, underserved areas and providing incentives for existing food retailers to offer healthier products. The economic feasibility of implementing these types of interventions depends on the policymakers ability to identify communities most at need. We use computer simulations, based on introducing new chain grocers in targeted areas, to map the effects on BMI of this modification in the food environment. In this study, we show that targeting economically disadvantaged communities with high prevalence of obesity-related diseases can provide an effective means of identifying areas where policy implementation will be most beneficial for improvements in health outcomes such as BMI.
A humanized IgG4 anti-HLA-DR monoclonal antibody (IMMU-114), engineered to avoid side effects associated with complement activation, was examined for binding and cytotoxicity on leukemia, lymphoma, and multiple myeloma cell lines and chronic lymphocytic leukemia (CLL) patient specimens, followed by evaluation of the effects of IMMU-114 on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. HLA-DR was expressed on the majority of these cells at markedly higher levels than CD20, CD22, and CD74. IMMU-114 was toxic to mantle cell lymphoma, CLL, acute lymphoblastic leukemia, hairy cell leukemia, non-Hodgkin lymphoma (including rituximab-resistant), and multiple myeloma cell lines, and also patient CLL cells. IMMU-114 induced disease-free survival in tumor-bearing SCID mice with early-stage disease and in models that are relatively resistant to anti-CD20 monoclonal antibodies. Despite positive staining, acute myelogenous leukemic cells were not killed by IMMU-114. The ability of IMMU-114 to induce activation of ERK and JNK signaling correlated with cytotoxicity and differentiates the mechanism of action of IMMU-114 from monoclonal antibodies against CD20 and CD74. Thus, antigen expression is not sufficient for cytotoxicity; antibody-induced hyperactivation of ERK and JNK mitogen activated protein kinase signaling pathways are also required.
Thermogenesis by resting muscle varies with conditions and plays an active role in homeostasis of body weight. The low metabolic rate of living resting muscles requires that ATP turnover by myosin be inhibited relative to the purified protein in vitro. This inhibition has not been previously seen in in vitro systems. We used quantitative epifluorescence microscopy of fluorescent nucleotides to measure single nucleotide turnovers in relaxed, permeable skeletal muscle fibers. We observed two lifetimes for nucleotide release by myosin: a fast component with a lifetime of approximately 20 s, similar to that of purified myosin, and a slower component with a lifetime of 230 +/- 24 s. We define the latter component to be the "super relaxed state." The fraction of myosins in the super relaxed state was decreased at lower temperatures, by substituting GTP for ATP or by increased levels of myosin phosphorylation. All of these conditions have also been shown to cause increased disorder in the structure of the thick filament. We propose a model in which the structure of the thick filament modulates the nucleotide turnover rates of myosin in relaxed fibers. Modulation of the relative populations of the super relaxed and conventional relaxed states could have a profound effect on muscle thermogenesis, with the capacity to also significantly alter whole-body metabolic rate.
The humanized anti-CD74 monoclonal antibody, milatuzumab, is in clinical evaluation for the therapy of multiple myeloma (MM). The ability of milatuzumab to increase the efficacy of bortezomib, doxorubicin, and dexamethasone was examined in three human CD74+ MM cell lines, CAG, KMS11, KMS12-PE, and one CD74-MM cell line, OPM-2.
This paper presents a new method of assessing the relationship between features of the built environment and obesity, particularly in urban areas. Our empirical application combines georeferenced data on the location of fast-food restaurants with data about personal health, behavioral, and neighborhood characteristics. We define a local food environment for every individual utilizing buffers around a persons home address. Individual food landscapes are potentially endogenous because of spatial sorting of the population and food outlets, and the body mass index (BMI) values for individuals living close to each other are likely to be spatially correlated because of observed and unobserved individual and neighborhood effects. The potential biases associated with endogeneity and spatial correlation are handled using spatial econometric estimation techniques. Our application provides quantitative estimates of the effect of proximity to fast-food restaurants on obesity in an urban food market. We also present estimates of a policy simulation that focuses on reducing the density of fast-food restaurants in urban areas. In the simulations, we account for spatial heterogeneity in both the policy instruments and individual neighborhoods and find a small effect for the hypothesized relationships between individual BMI values and the density of fast-food restaurants.
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