A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.     

Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult‐Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model

Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease‐related neuroimmune responses in the adult brain. Recently, an adult mouse brain‐derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S‐Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (≈300 000). Using a 2‐h gradient on a 75‐cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole‐Orbitrap mass spectrometer, 4855 total proteins have been identified where 4698 of which are quantifiable by label‐free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult‐derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006.     

Integrating a Portable Biofeedback Device into Clinical Practice for Patients with Anxiety Disorders: Results of a Pilot Study

This study examined the effectiveness of a portable Respiratory Sinus Arrhythmia (RSA) biofeedback device as an adjunct to CBT in persons with anxiety disorders and other disorders associated with autonomic dysfunction attending outpatient treatment. Participants were 24 individuals attending outpatient cognitive behavioral treatment for a range of anxiety disorders. Participants were assessed over a 3 week period. Outcomes included measures of anxiety (STAI-Y), sleep disturbances (PSQI), anger (STAEI), and subjective questions about the effectiveness of the device as a treatment adjunct. Significant reductions were found for anxiety and anger and for certain sleep variables (e.g. sleep latency). There was a significant dos–effect in that those who were more compliant had significantly greater reductions in most domains including sleep, anger and trait anxiety. Overall, participants found the device more helpful than other relaxation techniques such as mediation, yoga and unassisted breathing techniques but less helpful than exercise. The most frequently endorsed side effects were dizziness (15%) and sleepiness (55%). These preliminary results suggest that portable RSA biofeedback appears to be a promising treatment adjunct for disorders of autonomic arousal and is easily integrated into treatment. Results support the need for further investigation with more rigorous experimental designs. This study was conducted at the Behavioral Associates and The CBT Institute in New York, NY from 6/06 through 8/06.     

Screening for Chronic Obstructive Pulmonary Disease: Validity and Reliability of a Portable Device in Non-Specialized Healthcare Settings

Introduction and Objectives

The underdiagnosis of chronic obstructive pulmonary disease (COPD) could be improved through screening using portable devices simpler than conventional spirometers in specific healthcare settings to reach a higher percentage of the at-risk population. This study was designed to assess the validity and reliability of the COPD-6 portable device to screen for COPD in non-specialized healthcare settings.

Methods

Prospective cohort study to validate a diagnostic test. Three cohorts were recruited: primary care (PC), emergency services (ES) and community pharmacies (CPh). Study population: individuals with risk factors for COPD (>40 years, smoking >10 pack-years, with respiratory symptoms). The values measured using the COPD-6 were FEV1, FEV6 and the FEV1/FEV6 ratio. Subsequently, participants underwent conventional spirometry at hospital, using a post-bronchodilator FEV1/FVC value <0.7 as the gold standard criterion for the COPD diagnosis.

Results

437 participants were included, 362 were valid for the analysis. COPD was diagnosed in 114 patients (31.5%). The area under the ROC curve for the COPD-6 for COPD screening was 0.8.The best cut-off point for the FEV1/FEV6 ratio was 0.8 (sensitivity, 92.1%) using spirometry with the bronchodilator test as the gold standard. There were practically no differences in the COPD-6 performancein the different settings and also regarding age, gender and smoking status.

Conclusions

The COPD-6 device is a valid tool for COPD screening in non-specialized healthcare settings. In this context, the best cut-off point for the FEV1/FEV6 ratio is 0.8.     

Selection of Reliable Biomarkers from PCR Array Analyses Using Relative Distance Computational Model: Methodology and Proof-of-Concept Study

It is increasingly evident about the difficulty to monitor chemical exposure through biomarkers as almost all the biomarkers so far proposed are not specific for any individual chemical. In this proof-of-concept study, adult male zebrafish (Danio rerio) were exposed to 5 or 25 µg/L 17β-estradiol (E2), 100 µg/L lindane, 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 15 mg/L arsenic for 96 h, and the expression profiles of 59 genes involved in 7 pathways plus 2 well characterized biomarker genes, vtg1 (vitellogenin1) and cyp1a1 (cytochrome P450 1A1), were examined. Relative distance (RD) computational model was developed to screen favorable genes and generate appropriate gene sets for the differentiation of chemicals/concentrations selected. Our results demonstrated that the known biomarker genes were not always good candidates for the differentiation of pair of chemicals/concentrations, and other genes had higher potentials in some cases. Furthermore, the differentiation of 5 chemicals/concentrations examined were attainable using expression data of various gene sets, and the best combination was the set consisting of 50 genes; however, as few as two genes (e.g. vtg1 and hspa5 [heat shock protein 5]) were sufficient to differentiate the five chemical/concentration groups in the present test. These observations suggest that multi-parameter arrays should be more reliable for biomonitoring of chemical exposure than traditional biomarkers, and the RD computational model provides an effective tool for the selection of parameters and generation of parameter sets.     

电穿孔下离子型药物透皮扩散的一类数学模型及其建模方法论

在分子型模型的基础上,导出了离子型药物经皮渗透过程的一类数学模型,并用计算机模拟了其特性、而且针对分子型和离子型模型中,待估参数相对于实验样本点而言过多,同时需处理大批在不同实验控制条件下的数据资料的情形,讨论了其建模方法论问题。     

A Comparative Study of the Bactericidal Activity and Daily Disinfection Housekeeping Surfaces by a New Portable Pulsed UV Radiation Device

Daily cleaning and disinfecting of non-critical surfaces in the patient-care areas are known to reduce the occurrence of health care-associated infections. However, the conventional means for decontamination of housekeeping surfaces of sites of frequent hand contact such as manual disinfection using ethanol wipes are laborious and time-consuming in daily practice. This study evaluated a newly developed portable pulsed ultraviolet (UV) radiation device for its bactericidal activity in comparison with continuous UV-C, and investigated its effect on the labor burden when implemented in a hospital ward. Pseudomonas aeruginosa, Multidrug-resistant P. aeruginosa, Escherichia coli, Acinetobacter baumannii, Amikacin and Ciprofloxacin-resistant A. baumannii, Staphylococcus aureus, Methicillin-resistant S. aureus and Bacillus cereus were irradiated with pulsed UV or continuous UV-C. Pulsed UV and continuous UV-C required 5 and 30 s of irradiation, respectively, to attain bactericidal activity with more than 2Log growth inhibition of all the species. The use of pulsed UV in daily disinfection of housekeeping surfaces reduced the working hours by half in comparison to manual disinfection using ethanol wipes. The new portable pulsed UV radiation device was proven to have a bactericidal activity against critical nosocomial bacteria, including antimicrobial-resistant bacteria after short irradiation, and was thus found to be practical as a method for disinfecting housekeeping surfaces and decreasing the labor burden.     

转基因玉米种子快速筛查方法研究与应用

转基因种子快速筛查技术研究与应用对于加强农业转基因生物安全管理至关重要。将DNA热碱提取法和复合荧光PCR技术相结合,建立起玉米种子转基因成分快速筛查方法。结果显示,该方法不仅能保证检测的准确性和灵敏性,而且还大大降低假阳性发生率,提高转基因筛查的效率,适合大批量样品的快速检测。     

Acceptability of Using Electronic Vending Machines to Deliver Oral Rapid HIV Self-Testing Kits: A Qualitative Study

Introduction

Rates of unrecognized HIV infection are significantly higher among Latino and Black men who have sex with men (MSM). Policy makers have proposed that HIV self-testing kits and new methods for delivering self-testing could improve testing uptake among minority MSM. This study sought to conduct qualitative assessments with MSM of color to determine the acceptability of using electronic vending machines to dispense HIV self-testing kits.

Materials and Methods

African American and Latino MSM were recruited using a participant pool from an existing HIV prevention trial on Facebook. If participants expressed interest in using a vending machine to receive an HIV self-testing kit, they were emailed a 4-digit personal identification number (PIN) code to retrieve the test from the machine. We followed up with those who had tested to assess their willingness to participate in an interview about their experience.

Results

Twelve kits were dispensed and 8 interviews were conducted. In general, participants expressed that the vending machine was an acceptable HIV test delivery method due to its novelty and convenience.

Discussion

Acceptability of this delivery model for HIV testing kits was closely associated with three main factors: credibility, confidentiality, and convenience. Future research is needed to address issues, such as user-induced errors and costs, before scaling up the dispensing method.     

A Novel Model of Chronic Wounds: Importance of Redox Imbalance and Biofilm-Forming Bacteria for Establishment of Chronicity

Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone [1]. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.     

Quantification of the In Vitro Radiosensitivity of Mung Bean Sprout Elongation to 6MV X-Ray: A Revised Target Model Study

In this study, a revised target model for quantifying the in vitro radiosensitivity of mung bean sprout elongation to 6-MV X-rays was developed. The revised target model, which incorporated the Poisson prediction for a low probability of success, provided theoretical estimates that were highly consistent with the actual data measured in this study. The revised target model correlated different in vitro radiosensitivities to various effective target volumes and was successfully confirmed by exposing mung beans in various elongation states to various doses of 6-MV X-rays. For the experiment, 5,000 fresh mung beans were randomly distributed into 100 petri dishes, which were randomly divided into ten groups. Each group received an initial watering at a different time point prior to X-ray exposure, resulting in different effective target volumes. The bean sprouts were measured 70 hr after X-ray exposure, and the average length of the bean sprouts in each group was recorded as an index of the mung bean in vitro radiosensitivity. Mung beans that received an initial watering either six or sixteen hours before X-ray exposure had the shortest sprout length, indicating that the maximum effective target volume was formed within that specific time period. The revised target model could be also expanded to interpret the “two-hit” model of target theory, although the experimental data supported the “one-hit” model. If the “two-hit” model was sustained, theoretically, the target size would be 2.14 times larger than its original size to produce the same results.     

Application of Random Coefficient Regression Model to Myopia Data: A Case Study

A one-year birth cohort from Northern Finland has been followed up since 1966. As a part of this study, we are in this paper concerned with analysing the progression of myopia (nearsightness) up to the age of 20 years. The random coefficient regression model was chosen for the analysis because of the large individual variation in the development of myopia. Maximum likelihood estimates for the parameters in the model were obtained via the expectation maximization (EM) algorithm. It is shown how the estimated model can be used to predict future observations for an individual using the previously recorded refractive error measurements as well as other relevant data on the patient in question.     

A Preclinical Evaluation of Alternative Synthetic Biomaterials for Fascial Defect Repair Using a Rat Abdominal Hernia Model

Introduction

Fascial defects are a common problem in the abdominal wall and in the vagina leading to hernia or pelvic organ prolapse that requires mesh enhancement to reduce operation failure. However, the long-term outcome of synthetic mesh surgery may be unsatisfactory due to post-surgical complications. We hypothesized that mesh fabricated from alternative synthetic polymers may evoke a different tissue response, and provide more appropriate mechanical properties for hernia repair. Our aim was to compare the in vivo biocompatibility of new synthetic meshes with a commercial mesh.

Methods

We have fabricated 3 new warp-knitted synthetic meshes from different polymers with different tensile properties polyetheretherketone (PEEK), polyamide (PA) and a composite, gelatin coated PA (PA+G). The rat abdominal hernia model was used to implant the meshes (25×35 mm, n = 24/ group). After 7, 30, 60, 90 days tissues were explanted for immunohistochemical assessment of foreign body reaction and tissue integration, using CD31, CD45, CD68, alpha-SMA antibodies. The images were analysed using an image analysis software program. Biomechanical properties were uniaxially evaluated using an Instron Tensile® Tester.

Results

This study showed that the new meshes induced complex differences in the type of foreign body reaction over the time course of implantation. The PA, and particularly the composite PA+G meshes, evoked a milder early inflammatory response, and macrophages were apparent throughout the time course. Our meshes led to better tissue integration and new collagen deposition, particularly with the PA+G meshes, as well as greater and sustained neovascularisation compared with the PP meshes.

Conclusion

PA, PA+G and PEEK appear to be well tolerated and are biocompatible, evoking an overlapping and different host tissue response with time that might convey mechanical variations in the healing tissue. These new meshes comprising different polymers may provide an alternative option for future treatment of fascial defects.     

Validity and Reliability of Using a Self-Lavaging Device for Cytology and HPV Testing for Cervical Cancer Screening: Findings from a Pilot Study

Self-sampling could increase cervical cancer screening uptake. While methods have been identified for human papillomavirus (HPV) testing, to date, self-sampling has not provided adequate specimens for cytology. We piloted the validity and reliability of using a self-lavaging device for cervical cytology and HPV testing. We enrolled 198 women in New York City in 2008–2009 from three ambulatory clinics where they received cervical cancer screening. All were asked to use the Delphi Screener™ to self-lavage 1–3 months after clinician-collected index cytological smear (100 normal; 98 abnormal). Women with abnormal cytology results from either specimen underwent colposcopy; 10 women with normal results from both specimens also underwent colposcopy. We calculated sensitivity of self-collected cytology to detect histologically confirmed high grade lesions (cervical intraepithelial neoplasia, CIN, 2+); specificity for histology-negative (CIN 1 or lower), paired cytology negative, or a third cytology negative; and kappa for paired results. One hundred and ninety-seven (99.5%) women self-collected a lavage. Seventy-five percent had moderate to excellent cellularity, two specimens were unsatisfactory for cytology. Seven of 167 (4%) women with definitive results had CIN2+; one had normal and six abnormal cytology results with the self-lavage (sensitivity = 86%, 95% Confidence Interval, CI: 42, 100). The kappa for paired cytology was low (0.36; 95% CI: 0.25, 0.47) primarily due to clinician specimens with atypical squamous cells of undetermined significance (ASC-US) and low grade squamous intraepithelial lesion (LSIL) coded as normal using Screener specimens. However, three cases of HSIL were coded as ASC-US and one as normal using Screener specimens. Seventy-three women had paired high-risk HPV tests with a kappa of 0.66 (95% CI: 0.49, 0.84). Based on these preliminary findings, a larger study to estimate the performance of the Screener for co-testing cytology and HPV or for HPV testing with cytology triage is warranted.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00702208","term_id":"NCT00702208"}}NCT00702208     

A Semiparametric Joint Model for Longitudinal and Survival Data with Application to Hemodialysis Study

Summary .  In many longitudinal clinical studies, the level and progression rate of repeatedly measured biomarkers on each subject quantify the severity of the disease and that subject's susceptibility to progression of the disease. It is of scientific and clinical interest to relate such quantities to a later time-to-event clinical endpoint such as patient survival. This is usually done with a shared parameter model. In such models, the longitudinal biomarker data and the survival outcome of each subject are assumed to be conditionally independent given subject-level severity or susceptibility (also called frailty in statistical terms). In this article, we study the case where the conditional distribution of longitudinal data is modeled by a linear mixed-effect model, and the conditional distribution of the survival data is given by a Cox proportional hazard model. We allow unknown regression coefficients and time-dependent covariates in both models. The proposed estimators are maximizers of an exact correction to the joint log likelihood with the frailties eliminated as nuisance parameters, an idea that originated from correction of covariate measurement error in measurement error models. The corrected joint log likelihood is shown to be asymptotically concave and leads to consistent and asymptotically normal estimators. Unlike most published methods for joint modeling, the proposed estimation procedure does not rely on distributional assumptions of the frailties. The proposed method was studied in simulations and applied to a data set from the Hemodialysis Study.     

A Model of Evolutionary Base Substitutions and Its Application with Special Reference to Rapid Change of Pseudogenes

A model of evolutionary base substitutions that can incorporate different substitutional rates between the four bases and that takes into account unequal composition of bases in DNA sequences is proposed. Using this model, we derived formulae that enable us to estimate the evolutionary distances in terms of the number of nucleotide substitutions through comparative studies of nucleotide sequences. In order to check the validity of various formulae, Monte Carlo experiments were performed. These formulae were applied to analyze data on DNA sequences from diverse organisms. Particular attention was paid to problems concerning a globin pseudogene in the mouse and the time of its origin through duplication. We obtained a result suggesting that the evolutionary rates of substitution in the first and second codon positions of the pseudogene were roughly 10 times faster than those in the normal globin genes; whereas, the rate in the third position remained almost unchanged. Application of our formulae to histone genes H2B and H3 of the sea urchin showed that, in each of these genes, the rate in the third codon position is tremendously higher than that in the second position. All of these observations can easily and consistently be interpreted by the neutral theory of molecular evolution.     

A Pilot Study Combining a GC-Sensor Device with a Statistical Model for the Identification of Bladder Cancer from Urine Headspace

There is a need to reduce the number of cystoscopies on patients with haematuria. Presently there are no reliable biomarkers to screen for bladder cancer. In this paper, we evaluate a new simple in–house fabricated, GC-sensor device in the diagnosis of bladder cancer based on volatiles. Sensor outputs from 98 urine samples were used to build and test diagnostic models. Samples were taken from 24 patients with transitional (urothelial) cell carcinoma (age 27-91 years, median 71 years) and 74 controls presenting with urological symptoms, but without a urological malignancy (age 29-86 years, median 64 years); results were analysed using two statistical approaches to assess the robustness of the methodology. A two-group linear discriminant analysis method using a total of 9 time points (which equates to 9 biomarkers) correctly assigned 24/24 (100%) of cancer cases and 70/74 (94.6%) controls. Under leave-one-out cross-validation 23/24 (95.8%) of cancer cases were correctly predicted with 69/74 (93.2%) of controls. For partial least squares discriminant analysis, the correct leave-one-out cross-validation prediction values were 95.8% (cancer cases) and 94.6% (controls). These data are an improvement on those reported by other groups studying headspace gases and also superior to current clinical techniques. This new device shows potential for the diagnosis of bladder cancer, but the data must be reproduced in a larger study.