Bioconjugated graphene oxide-based Raman probe for selective identification of SKBR3 breast cancer cells.

In this article, we demonstrate the use of bio-conjugated 2D graphene oxide (bio-GO) nanostructure to probe breast cancer cell (SKBR3) with excellent discrimination over other types of circulating tumor cells. We distinctly observed that bio-GO nanostructure targets and bind SKBR3 cell selectively in the cell mixture. Longer incubation of SKBR3 cell with bio-GO causes Raman signal "turn off" when excited with 532 nm laser. This is attributed to penetration of the bio-GO through the plasma membrane of the cell by generating transient hole. Extraction of GO after cell digestion also support the internalization rubric of 2D graphene through cell membrane. Our experimental data with the HaCaT healthy cell line, as well as with LNCaP prostate cancer cell line clearly demonstrated that this Raman scattering assay is highly selective to SKBR3. The mechanism of selectivity and the assay's response change have been verified and discussed utilizing fluorescence properties of GO and various other techniques. The experimental results open up a possibility of new label free Raman scattering assay, for reliable diagnosis of cancer cell lines by monitoring "turn-off" of the Raman signal from Bio-GO nanostructure.

Developing Measures of Pathways that May Link Macro Social/Structural Changes with HIV Epidemiology.

Macro-social/structural events ("big events") such as wars, disasters, and large-scale changes in policies can affect HIV transmission by making risk behaviors more or less likely or by changing risk contexts. The purpose of this study was to develop new measures to investigate hypothesized pathways between macro-social changes and HIV transmission. We developed novel scales and indexes focused on topics including norms about sex and drug injecting under different conditions, involvement with social groups, helping others, and experiencing denial of dignity. We collected data from 300 people who inject drugs in New York City during 2012-2013. Most investigational measures showed evidence of validity (Pearson correlations with criterion variables range = 0.12-0.71) and reliability (Cronbach's alpha range = 0.62-0.91). Research is needed in different contexts to evaluate whether these measures can be used to better understand HIV outbreaks and help improve social/structural HIV prevention intervention programs.

Measuring Altruistic and Solidaristic Orientations Toward Others Among People Who Inject Drugs.

The altruism and/or solidarity of people who inject drugs helps protect sex and drug partners from HIV. Research has been hindered by lack of measures. We developed and administered scales to assess them to 300 people who inject drugs. Altruism and Solidarity Scales were both internally consistent. Each correlated significantly with measures of helping others. These measures appear reliable and valid. They can be used to study how big events or structural interventions affect altruism and solidarity, and how altruism and solidarity are associated with changes in HIV or other risks, among people who inject drugs.

Formal and informal organizational activities of people who inject drugs in New York City: description and correlates.

Little is known about group memberships of people who inject drugs (PWID). Three hundred PWID were interviewed about formal and informal group participation and risk behaviors. Many took part in groups related to problems and resources associated with injecting drugs, religion, sports or gender. Harm reduction group and support group participation was associated with less risk behavior; sports groups participation with more risk behavior. Group involvement by PWID may be important to their lives and/or affect prevention or infectious disease transmission. More research is needed about determinants and consequences of their and other drug users' group memberships.

Identification of potential biomarkers of P-glycoprotein substrate neurotoxicity in transgenic mice expressing the mutated canine ABCB1 gene.

OBJECTIVE: To identify biomarkers of P-glycoprotein (P-gp) substrate neurotoxicity in transgenic mice expressing the mutant canine ABCB1 gene (ABCB1-1Δ). ANIMALS: 8 ABCB1 knock-in and knock-out transgenic mice expressing the ABCB1-1Δ gene and 8 control mice expressing the wild-type canine ABCB1 gene (ABCB1-WT). PROCEDURES: Groups including 2 ABCB1-1Δ mutant mice and 2 ABCB1-WT mice were administered the P-gp substrates ivermectin (10 mg/kg, SC), doramectin (10 mg/kg, SC), moxidectin (10 mg/kg, PO), or digoxin (1.53 mg/kg, SC). A toxicogenomic approach based on DNA microarrays was used to examine whole-genome expression changes in mice administered P-gp substrates. RESULTS: Compared with control ABCB1-WT mice, ABCB1-1Δ mutant mice developed neurotoxic signs including ataxia, lethargy, and tremors similar to those reported for dogs with the ABCB1-1Δ mutation. Microarray analysis revealed that gene expression was altered in ABCB1-1Δ mutant mice, compared with findings for ABCB1-WT mice, following administration of the same P-gp substrates. Gene pathway analysis revealed that genes with a ≥ 2-fold gene expression change were associated with behavior and nervous system development and function. Moreover, 34 genes were altered in the ABCB1-1Δ mutant mice in all 4 drug treatment groups. These genes were also associated with behavior, which was identified as the top-ranked gene network. CONCLUSIONS AND CLINICAL RELEVANCE: These study data have facilitated understanding of the molecular mechanisms of neurotoxicosis in ABCB1-1Δ mutant mice following exposure to various P-gp substrates. Some genes appear to be potential biomarkers of P-gp substrate neurotoxicity that might be used to predict the safety of those drugs in dogs with the ABCB1-1Δ mutation.

Enhancing targeted tumor treatment by near IR light-activatable photodynamic-photothermal synergistic therapy.

For several decades, cancer has been one of the most life-threatening diseases. For enhancing anticancer efficiency with minimum side effects, combination therapy is envisioned. The current manuscript reports for the first time the development of a methylene blue (MB) bound nanoplatform, which is capable of delivering targeted diagnostic and combined synergistic photothermal and photodynamic treatment of cancer. Experimental data found that, once the nanoparticle binds with the target cell surface, it can detect LNCaP human prostate cancer cell selectively using fluorescence imaging. Our result shows that the therapeutic actions can be controlled with external NIR light. No cytotoxicity was observed in the absence of NIR light. Targeted photodynamic and photothermal treatment using 785 nm NIR light indicates that the multimodal treatment enhances the possibility of destroying LNCaP prostate cancer cells in vitro dramatically. We discuss the operating principle for the targeted imaging and possible mechanisms for combined therapeutic actions. Our experimental data show that NIR light activated combined therapy for cancer may become a highly effective treatment procedure in clinical settings.

Potential use of DNA barcodes in regulatory science: identification of the U.S. Food and Drug Administration's "Dirty 22," contributors to the spread of foodborne pathogens.

The U.S. Food, Drug, and Cosmetic Act prohibits the distribution of food that is adulterated, and the regulatory mission of the U.S. Food and Drug Administration (FDA) is to enforce this Act. FDA field laboratories have identified the 22 most common pests that contribute to the spread of foodborne disease (the "Dirty 22"). The current method of detecting filth and extraneous material (tails, legs, carcasses, etc.) is visual inspection using microscopy. Because microscopy can be time-consuming and may yield inaccurate and/or nonspecific results due to lack of expertise, an alternative method of detecting these adulterants is needed. In this study, we sequenced DNA from the 5' region of the cytochrome oxidase I gene of these 22 common pests that contribute to the spread of foodborne pathogens. Here, we describe the generation of DNA barcodes for all 22 species. To date, this is the first attempt to develop a sequence-based regulatory database and systematic primer strategy to identify these FDA-targeted species. DNA barcoding can be a powerful tool that can aid the FDA in promoting the protection and safety of the U.S. food supply.

Neurotoxic effects of ivermectin administration in genetically engineered mice with targeted insertion of the mutated canine ABCB1 gene.

OBJECTIVE: To develop in genetically engineered mice an alternative screening method for evaluation of P-glycoprotein substrate toxicosis in ivermectin-sensitive Collies. ANIMALS: 14 wild-type C57BL/6J mice (controls) and 21 genetically engineered mice in which the abcb1a and abcb1b genes were disrupted and the mutated canine ABCB1 gene was inserted. PROCEDURES: Mice were allocated to receive 10 mg of ivermectin/kg via SC injection (n = 30) or a vehicle-only formulation of propylene glycol and glycerol formal (5). Each was observed for clinical signs of toxic effects from 0 to 7 hours following drug administration. RESULTS: After ivermectin administration, considerable differences were observed in drug sensitivity between the 2 types of mice. The genetically engineered mice with the mutated canine ABCB1 gene had signs of severe sensitivity to ivermectin, characterized by progressive lethargy, ataxia, and tremors, whereas the wild-type control mice developed no remarkable effects related to the ivermectin. CONCLUSIONS AND CLINICAL RELEVANCE: The ivermectin sensitivity modeled in the transgenic mice closely resembled the lethargy, stupor, disorientation, and loss of coordination observed in ivermectin-sensitive Collies with the ABCB1-1Δ mutation. As such, the model has the potential to facilitate toxicity assessments of certain drugs for dogs that are P-glycoprotein substrates, and it may serve to reduce the use of dogs in avermectin derivative safety studies that are part of the new animal drug approval process.

Development of a multiplex real-time PCR assay for the detection of ruminant DNA.

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.

In vivo characterization of inflammatory biomarkers in swine and the impact of flunixin meglumine administration.

Non-steroidal anti-inflammatory drugs (NSAID) are a family of chemicals that function to reduce pain, fever, and inflammation, and they are commonly used in people and animals for this purpose. Currently there are no NSAIDs approved for the management of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess efficacy. A previous in vitro study examining biomarkers of inflammation identified fourteen genes that were significantly altered in response to Escherichia coli lipopolysaccharide (LPS)-induced inflammation. In the present study, five of those fourteen genes were tested in vivo to determine if the same effects observed in vitro were also observed in vivo. Plasma levels of prostaglandin E(2) (PGE(2)), an essential mediator of fever and inflammation, were also determined. Two groups of swine were stimulated with LPS with the second group also treated with flunixin meglumine. Blood was collected at 0, 1, 3, 6, 8, 24, and 48 h post LPS-stimulation. The RNA was extracted from the blood and quantitative real-time-PCR (qRT-PCR) was utilized to determine the expression patterns of CD1, CD4, serum amyloid A2 (SAA2), Caspase 1, and monocyte chemoattractant protein 1 (MCP-1). The LPS-stimulated animals demonstrated a statistically significant alteration in expression of SAA2 and CD1 at 3h post-stimulation. Flunixin meglumine treated animals' demonstrated reduced expression of CD1 in comparison to the LPS-stimulated swine at 24 and 48 h post LPS-stimulation. Flunixin meglumine treated animals exhibited reduced expression of SAA2 at 48 h post-stimulation compared to LPS-stimulated swine. Swine treated with LPS demonstrated statistically significant increases in plasma PGE(2) at 1h post-stimulation. Swine treated with flunixin meglumine had no increase in plasma PGE(2) levels at any time. These results demonstrate that PGE(2) production, along with two out of five genes (SAA2 and CD1) have the potential to serve as early biomarkers of inflammation as well as indicators of NSAID efficacy.

Real-time PCR assay for the detection of pufferfish products.

An assay was developed for the rapid detection of products containing tissues from potentially toxic pufferfish (family Tetraodontidae), as part of the U.S. Food and Drug Administration Center for Veterinary Medicine and Center for Food Safety and Applied Nutrition's charter to protect human health. In this study, we developed a TaqMan assay derived from DNA barcode data (650 bp starting at the 5' end of the mitochondrial cytochrome c oxidase I gene) for the specific detection of pufferfish. The method requires only 1 h of total run time, a significant improvement over current methods, which can require 24 to 96 h for completion. The probes were tested against 105 species of fish and were able to detect 20 species of pufferfish; no cross-reactivity was shown with 85 species of nonpufferfish, including 20 related species from the same order (Tetraodontiformes). These results demonstrate that this assay is suitable for the rapid and specific detection of pufferfish and that it could be a useful regulatory tool to protect human health.

Analysis of liquid-phase chemical detection using guided shear horizontal-surface acoustic wave sensors.

Direct chemical sensing in liquid environments using polymer-guided shear horizontal surface acoustic wave sensor platforms on 36 degrees rotated Y-cut LiTaO3 is investigated. Design considerations for optimizing these devices for liquid-phase detection are systematically explored. Two different sensor geometries are experimentally and theoretically analyzed. Dual delay line devices are used with a reference line coated with poly (methyl methacrylate) (PMMA) and a sensing line coated with a chemically sensitive polymer, which acts as both a guiding layer and a sensing layer or with a PMMA waveguide and a chemically sensitive polymer. Results show the three-layer model provides higher sensitivity than the four-layer model. Contributions from mass loading and coating viscoelasticity changes to the sensor response are evaluated, taking into account the added mass, swelling, and plasticization. Chemically sensitive polymers are investigated in the detection of low concentrations (1-60 ppm) of toluene, ethylbenzene, and xylenes in water. A low-ppb level detection limit is estimated from the present experimental measurements. Sensor properties are investigated by varying the sensor geometries, coating thickness combinations, coating properties, and curing temperature for operation in liquid environments. Partition coefficients for polymer-aqueous analyte pairs are used to explain the observed trend in sensitivity for the polymers PMMA, poly(isobutylene), poly(epichlorohydrin), and poly(ethyl acrylate) used in this work.

Counseling Latina mothers of preschool children about weight issues: suggestions for a new framework.

OBJECTIVE: To assess Latina mothers' health beliefs and attitudes regarding early childhood weight issues and to use the information to update current nutrition education methods. DESIGN: Data were collected in eight focus group sessions using a semistructured questionnaire. SUBJECTS/SETTING: Forty-three Latina mothers (and grandmothers) with children aged 2 to 5 years were recruited at five different Special Supplemental Nutrition Program for Women, Infants, and Children sites in California. ANALYSIS: Transcripts of focus groups were imported into QSR NUD*IST software, facilitating in-depth iterative analysis of emergent themes. RESULTS: Fifteen emergent themes were identified and organized into four functional domains relevant to nutrition education: health beliefs surrounding weight, impact and cause of overweight, life values and concerns, and strategies for making changes in children's eating and activity patterns. Information from this qualitative study demonstrates that the traditional nutrition counseling paradigm may not be effective with Latina mothers. In addition, cultural beliefs can be barriers to successful prevention and treatment of overweight. To ensure that culturally competent services are provided, educators must be prepared to adjust education approaches according to the cultural background of the clients. Key among the issues was mothers' difficulty acknowledging overweight among their children and their perception that health and weight were poorly associated. Certain cultural values were identified as barriers to adopting healthful behaviors. Mothers were able to identify specific ways in which nutrition education could be improved. APPLICATIONS/CONCLUSIONS: Our findings suggest that nutrition education efforts targeting Latina mothers of young children can be reframed to better address the belief system and cultural framework of the population, like identifying positive eating behaviors rather than focusing on a child's weight.