Search

Salmonella enterica serovar Enteritidis is one of the most frequent causes of Salmonellosis globally and is commonly transmitted from animals to humans by the consumption of contaminated foodstuffs. Herein, we detail the development and application of a hierarchical machine learning model to rapidly identify and trace the geographical source of S. Enteritidis infections from whole genome sequencing data. 2,313 S. Enteritidis genomes collected by the UKHSA between 2014-2019 were used to train a ‘local classifier per node’ hierarchical classifier to attribute isolates to 4 continents, 11 sub-regions and 38 countries (53 classes). Highest classification accuracy was achieved at the continental level followed by the sub-regional and country levels (macro F1: 0.954, 0.718, 0.661 respectively). A number of countries commonly visited by UK travellers were predicted with high accuracy (hF1: >0.9). Longitudinal analysis and validation with publicly accessible international samples indicated that predictions were robust to prospective external datasets. The hierarchical machine learning framework provides granular geographical source prediction directly from sequencing reads in

Surface enhanced Raman spectroscopy enables robust, rapid analysis on highly dilute samples. To be useful, the technique needs sensing substrates that will enhance intrinsically weak Raman signals of trace analytes. In particular, three-dimensional substrates such as zinc oxide nanowires decorated with electron-beam deposited silver nanoparticles are easily fabricated and serve the dual need of structural stability and detection sensitivity. However, little has been done to optimize electron beam-deposited silver nanoparticles for maximal surface enhancement in the unique dielectric environment of the zinc oxide substrate. Herein, fabrication and anneal parameters of electron beam-deposited silver nanoparticles were examined for the purpose of maximizing surface enhancement. Specifically, this work explored the effect of changing film thickness, deposition rate, anneal temperature, and anneal time on the surface plasmon resonance of Ag nanoparticles. In this study, multiple sets of fabrication and annealing parameters were discovered that optimized surface plasmon resonance for maximal enhancement to Raman signals acquired with a 532 nm laser. This work represents the first characterization of the fabrication and annealing parameters for electron beam-deposited silver nanoparticles on zinc oxide.

Shigellosis in men who have sex with men (MSM) is caused by multidrug resistant Shigellae, exhibiting resistance to antimicrobials including azithromycin, ciprofloxacin and more recently the third-generation cephalosporins. We sequenced four bla CTX-M-27-positive MSM Shigella isolates (2018–20) using Oxford Nanopore Technologies; three S. sonnei (identified as two MSM clade 2, one MSM clade 5) and one S. flexneri 3a, to explore AMR context. All S. sonnei isolates harboured Tn7/Int2 chromosomal integrons, whereas S. flexneri 3a contained the Shigella Resistance Locus. All strains harboured IncFII pKSR100-like plasmids (67-83kbp); where present bla CTX-M-27 was located on these plasmids flanked by IS26 and IS903B, however bla CTX-M-27 was lost in S. flexneri 3a during storage between Illumina and Nanopore sequencing. IncFII AMR regions were mosaic and likely reorganised by IS26; three of the four plasmids contained azithromycin-resistance genes erm(B) and mph(A) and one harboured the pKSR100 integron. Additionally, all S. sonnei isolates possessed a large IncB/O/K/Z plasmid, two of which carried aph(3’)-Ib/aph(6)-Id/sul2 and tet(A). Monitoring the transmission of mobile genetic elements with co-located AMR determinants is necessary to inform empirical treatment guidance and clinical management of MSM-associated shigellosis.

Infrared neural stimulation, or INS, is a method of using pulsed infrared light to yield label-free neural stimulation with broad experimental and translational utility. Despite its robust demonstration, the mechanistic and biophysical underpinnings of INS have been the subject of debate for more than a decade. The role of lipid membrane thermodynamics appears to play an important role in how fast IR-mediated heating nonspecifically drives action potential generation. Direct observation of lipid membrane dynamics during INS remains to be shown in a live neural model system. To directly test the involvement of lipid dynamics in INS, we used hyperspectral stimulated Raman scattering (hsSRS) microscopy to study biochemical signatures of high-speed vibrational dynamics underlying INS in a live neural cell culture model. Findings suggest that lipid bilayer structural changes are occurring during INS in vitro in NG108-15 neuroglioma cells. Lipid-specific signatures of cell SRS spectra were found to vary with stimulation energy and radiant exposure. Spectroscopic observations were verified against high-speed ratiometric fluorescence imaging of a conventional lipophilic membrane structure reporter, di-4-ANNEPS. Overall, the presented data supports the hypothesis that INS causes changes in the lipid membrane of neural cells by changing lipid membrane packing order – which coincides with likelihood of cell stimulation. Furthermore, this work highlights the potential of hsSRS as a method to study biophysical and biochemical dynamics safely in live cells.

Otitis media (OM) is a group of inflammatory diseases of the middle ear and is the most frequent cause of physician visits and antibiotic prescriptions for children. Current diagnostic methods do not differentiate acute otitis media from otitis media with effusion, as both types of OM share many symptomatic features. A cohort of around 50 patients undergoing myringotomy were enrolled in this study, where Raman spectroscopy (RS) allowed non-invasive determination of OM subtype. Here we demonstrate the potential for RS to probe OM infection status in pediatric patients, to guide treatment protocol for ear infections and limit over-prescription of antibiotics.

Current diagnosis of pediatric eosinophilic esophagitis relies heavily on anesthesia and random biopsies during initial diagnosis and treatment monitoring. Investigation of patient’s saliva biochemical and biomolecular composition, and the potential biomarkers within, via surface enhanced Raman spectroscopy can provide key markers for distinguishing the EoE disease from non-EoE, including acid reflux disease. This optical modality coupled with paper-fluidic platform has the potential to provide a rapid diagnostic tool for non-invasive detection of EoE.

Bacterial infection is a global burden that results in numerous hospital visits and deaths annually. The rise of multi-drug resistant bacteria has dramatically increased this burden. Therefore, there is a clinical need to detect and identify bacteria rapidly and accurately in their native state or a culture-free environment. Current diagnostic techniques lack speed and effectiveness in detecting bacteria that are culture-negative, as well as options for in vivo detection. The optical detection of bacteria offers the potential to overcome these obstacles by providing various platforms that can detect bacteria rapidly, with minimum sample preparation, and, in some cases, culture-free directly from patient fluids or even in vivo. These modalities include infrared, Raman, and fluorescence spectroscopy, along with optical coherence tomography, interference, polarization, and laser speckle. However, these techniques are not without their own set of limitations. This review summarizes the strengths and weaknesses of utilizing each of these optical tools for rapid bacteria detection and identification.

Towards achieveing a subcutaneously implanted glucose biosensor with long-term functionality, a thermoresponsive membrane previously shown to have potential to house a glucose sensing assay was evaluated herein for its ability to minimize the foriegn body reaction (FBR) and the resulting fibrous capsule. The severity of the FBR proportionally reduces diffusion of glucose to the sensor and hence sensor lifetime. However, efforts to reduce the FBR have largedly focused on anti-fouling materials that passively inhibit cellular attachment, particularly poly(ethylene glycol) (PEG). Herein, the extent of the FBR of a subcutaneously implanted "self-cleaning" cylindrical membrane was analyzed in rodents. This membrane represents an "actively anti-fouling" approach to reduce cellular adhesion. It is a thermoresponsive double network nanocomposite hydrogel (DNNC) comprised of poly(N-isopropylacrylamide) (PNIPAAm) and embedded polysiloxane nanoparticles. The membrane's cyclical deswelling/reswelling response to local body temperature fluctuations was anticipated to limit cellular accumulation. Indeed, after 30 days, the self-cleaning membrane exhibited a notably thin fibrous capsule (~30 µm) and increased microvascular density within 1 mm of the implant surface in comparison to a non-thermoresponsive, benchmark biocompatible control (PEG diacrylate, PEG-DA).

Point-of-care (POC) device development is a growing field that aims to develop low-cost, rapid, sensitivein-vitrodiagnostic testing platforms that are portable, self-contained, and can be used anywhere – from modern clinics to remote and low resource areas. In this review, surface enhanced Raman spectroscopy (SERS) is discussed as a solution to facilitating the translation of bioanalytical sensing to the POC. The potential for SERS to meet the widely accepted “ASSURED” (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable) criterion provided by the World Health Organization is discussed based on recent advances in SERSin vitroassay development. As SERS provides attractive characteristics for multiplexed sensing at low concentration limits with a high degree of specificity, it holds great promise for enhancing current efforts in rapid diagnostic testing. In outlining the progression of SERS techniques over the past years combined with recent developments in smart nanomaterials, high-throughput microfluidics, and low-cost paper diagnostics, an extensive number of new possibilities show potential for translating SERS biosensors to the POC.

The focus of this work is toward the development of a point-of-care (POC) handheld technology for the noninvasive early detection of salivary biomarkers. The initial of focus was the detection and quantification of S100 calcium-binding protein P (S100P) mRNA found in whole saliva for use as a potential biomarker for oral cancer. Specifically, a surface-enhanced Raman spectroscopy (SERS)-based approach and assay were designed, developed, and tested for sensitive and rapid detection of S100P mRNA. Gold nanoparticles (AuNPs) were conjugated with oligonucleotides and malachite green isothiocyanate was then used as a Raman reporter molecule. The hybridization of S100P target to DNA-conjugated AuNPs in sandwich assay format in both free solution and a vertical flow chip (VFC) was confirmed using a handheld SERS system. The detection limit of the SERS-based assay in free solution was determined to be 1.1 nM, whereas on the VFC the detection limit was observed to be 10 nM. SERS-based VFCs were also used to quantify the S100P mRNA from saliva samples of oral cancer patients and a healthy group. The result indicated that the amount of S100P mRNA detected for the oral cancer patients is three times higher than that of a healthy group.

Esophageal diseases result in significant mortality, morbidity, and health care costs worldwide. Current approaches to detect and monitor esophageal diseases have severe limitations. Advanced imaging technologies are being developed to complement current approaches to improve diagnostic, therapeutic and surveillance protocols in order to advance the field. Raman spectroscopy-based technologies hold promise to increase the sensitivity for detection of diseased and high-risk lesions in vitro and in vivo in real time. This technique allows for the investigation of microstructural changes and also facilitates the discovery of disease-specific biochemical alterations with the potential to provide novel insights into the pathobiology of these conditions. Raman spectroscopy has been increasingly applied in precancerous and cancerous esophageal conditions. However, its application in benign esophageal diseases is still in the early stages. Continuing its application in cancerous and precancerous conditions and expanding its use to benign esophageal disorders could lay a foundation for integration of this technology in clinical practice and diagnostic paradigms and development of an accurate and cost-effective tool for use in a clinical setting. Furthermore, Raman spectroscopy can also be used as an innovative technique to advance our understanding of the biochemical transformations associated with esophageal diseases and answer a myriad of fundamental questions in the field. In this review, we described the principles of Raman spectroscopy and instrumentation while providing an overview of current applications, challenges, and future directions in the context of esophageal diseases with an emphasis on its clinical translational application.

A continuous glucose monitoring device that resides fully in the subcutaneous tissue has the potential to greatly improve the management of diabetes. Toward this goal, we have developed a competitive binding glucose sensing assay based on fluorescently labeled PEGylated concanavalin-A (PEGylated-TRITC-ConA) and mannotetraose (APTS-MT). In the present work, we sought to contain this assay within the hollow central cavity of a cylindrical hydrogel membrane, permitting eventual subcutaneous implantation and optical probing through the skin. A "self-cleaning" hydrogel was utilized because of its ability to cyclically deswell/reswell in vivo, which is expected to reduce biofouling and therefore extend the sensor lifetime. Thus, we prepared a hollow, cylindrical hydrogel based on a thermoresponsive electrostatic double network design composed of N-isopropylacrylamide and 2-acrylamido-2-methylpropanesulfonic acid. Next, a layer-by-layer (LbL) coating was applied to the inner wall of the central cavity of the cylindrical membrane. It consisted of 5, 10, 15, 30, or 40 alternating bilayers of positively charged poly(diallyldimethylammonium chloride) and negatively charged poly(sodium 4-styrenesulfonate). With 30 bilayers, the leaching of the smaller-sized component of the assay (APTS-MT) from the membrane cavity was substantially reduced. Moreover, this LbL coating maintained glucose diffusion across the hydrogel membrane. In terms of sensor functionality, the assay housed in the hydrogel membrane cavity tracked changes in glucose concentration (0 to 600 mg/dL) with a mean absolute relative difference of ∼11%.

Research toward development of point-of-care (POC) technologies is emerging as a means for diagnosis and monitoring of patients outside the hospital. These POC devices typically utilize assays capable of detecting low level biomarkers indicative of specific diseases. L-citrulline, an α-amino acid produced in the intestinal mucosa cells, is one such biomarker typically found circulating within the plasma at physiological concentrations of ~40 μM. Researchers have found that intestinal enterocyte malfunction causes its level to be significantly lowered, establishing it as a potential diagnostic biomarker for gut function. Our research group has proposed the development of a surface enhanced Raman spectroscopy (SERS) based assay, using vertical flow paper fluidics, for citrulline detection. The assay consists of a fluorescently active, Raman reporter labeled aptamer conjugated on gold nanoparticles. The aptamer changes its confirmation on binding to its target, which in turn changes the distance between the Raman active molecule and the nanoparticle surface. These particles were embedded within a portable chip consisting of cellulose-based paper. After the chips were loaded with different concentrations of free L-citrulline in phosphate buffer, time was given for the assay to interact with the sample. A handheld Raman spectrometer (638 nm; Ocean Optics) was used to measure the SERS intensity. Results showed decrease in intensity with increasing concentration of L-citrulline (0-50μM).

Metal nanoparticles conjugated with DNA oligomers have been intensively studied for a variety of applications, including optical diagnostics. Assays based on aggregation of DNA-coated particles in proportion to the concentration of target analyte have not been widely adopted for clinical analysis, however, largely due to the nonspecific responses observed in complex biofluids. While sample pre-preparation such as dialysis is helpful to enable selective sensing, here we sought to prove that assay encapsulation in hollow microcapsules could remove this requirement and thereby facilitate more rapid analysis on complex samples. Gold nanoparticle-based assays were incorporated into capsules comprising polyelectrolyte multilayer (PEMs), and the response to small molecule targets and larger proteins were compared. Gold nanoparticles were able to selectively sense small Raman dyes (Rhodamine 6G) in the presence of large protein molecules (BSA) when encapsulated. A ratiometric based microRNA-17 sensing assay exhibited drastic reduction in response after encapsulation, with statistically-significant relative Raman intensity changes only at a microRNA-17 concentration of 10 nM compared to a range of 0-500 nM for the corresponding solution-phase response.

It is estimated that the number of new cases of oral cancers worldwide is 529,000 and more than 300,000 deaths each year. The five-year survival rate remains about 50%, and the low survival rate is believed to be due to delayed detection. The primary detection method is through a comprehensive clinical examination by a dentist followed by a biopsy of suspicious lesions. Systematic review and meta-analysis have revealed that clinical examination alone may not be sufficient to cause the clinician to perform a biopsy or refer for biopsy for early detection of OSCC. Therefore, a non-invasive, point-of-Care (POC) detection with high sensitivity and specificity for early detection would be urgently needed, and using salivary biomarkers would be an ideal technology for it. S100 calcium binding protein P (S100P) mRNA presenting in saliva is a potential biomarker for detection of oral cancer. Further, surface enhanced Raman spectroscopy (SERS) has been shown to be a promising POC diagnostic technique. In this research, a SERS-based assay using oligonucleotide strains was developed for the sensitive and rapid detection of S100P. Gold nanoparticles (AuNPs) as a SERS substrate were used for the conjugation with one of two unique 24 base pair oligonucleotides, referred to as left and right DNA probes. A Raman reporter molecule, malachite green isothiocyanate (MGITC), was bound to left-probe-conjugated AuNPs. UV-vis spectroscopy was employed to monitor the conjugation of DNA probes to AuNPs. The hybridization of S100P target to DNA-conjugated AuNPs in sandwich-assay format was confirmed by Raman spectroscopy and shown to yield and R2 of 0.917 across the range of 0-200 nM and a limit of detection of 3 nM.

Competitive binding assays based on the lectin Concanavalin A (ConA) have displayed significant potential to serve in continuous glucose monitoring applications. However, to date, this type of fluorescent, affinity-based assay has yet to show the stable, glucose predictive capabilities that are required for such an application. This instability has been associated with the extensive crosslinking between traditionally-used fluorescent ligands (presenting multiple low-affinity moieties) and ConA (presenting multiple binding sites) in free solution. The work herein introduces the design and synthesis of a new type of fluorescent ligand that can avoid this aggregation and allow the assay to be sensitive across the physiologically relevant glucose concentration range. This fluorescent ligand (APTS-MT) presents a single high-affinity trimannose moiety that is recognized by ConA’s full binding site and a fluorophore that can effectively track the ligand’s equilibrium binding via fluorescent anisotropy. This is confirmed by comparing its measured fluorescent lifetime to experimentally-determined rotational correlation lifetimes of the free and bound populations. Using an assay comprised of 200 nM APTS-MT and 1 μM ConA, the fluorescence anisotropy capably tracks the concentration of monosaccharides that are known to bind to ConA’s primary binding site, and the assay displays a MARD of 6.5% across physiologically relevant glucose concentrations. Ultimately, this rationally-designed fluorescent ligand can facilitate the realization of the full potential of ConA-based glucose sensing assays and provide the basis for a new set of competing ligands to be paired with ConA.

A self-cleaning membrane that periodically rids itself of attached cells to maintain glucose diffusion could extend the lifetime of implanted glucose biosensors. Herein, we evaluate the functionality of thermoresponsive double network (DN) hydrogel membranes based on poly(N-isopropylacrylamide) (PNIPAAm) and an electrostatic co-monomer, 2-acrylamido-2-methylpropane sulfonic acid (AMPS). DN hydrogels are comprised of a tightly crosslinked, ionized first network [P(NIPAAm-co-AMPS)] containing variable levels of AMPS (100:0-25:75 wt% ratio of NIPAAm:AMPS) and a loosely crosslinked, interpenetrating second network [PNIPAAm]. To meet the specific requirements of a subcutaneously implanted glucose biosensor, the volume phase transition temperature is tuned and essential properties, such as glucose diffusion kinetics, thermosensitivity, and cytocompatibility are evaluated. In addition, the self-cleaning functionality is demonstrated through thermally driven cell detachment from the membranes in vitro.

Diabetes mellitus affects millions of people worldwide and requires that individuals tightly self-regulate their blood glucose levels to minimize the associated secondary complications. Continuous monitoring devices potentially offer patients a long-term means to tightly monitor their glucose levels. In recent years, fluorescent affinity sensors based on lectins (e.g., Concanavalin A (ConA)) have been implemented in such devices. Traditionally, these sensors pair the lectin with a multivalent ligand, like dextran, in order to develop a competitive binding assay that changes its fluorescent properties in response to the surrounding glucose concentrations. This work introduces a new type of fluorescent ligand for FRET-based assays in an attempt to improve the sensitivity of such assays. This ligand is rationally designed to present a core trimannose structure and a donor fluorophore in close proximity to one another. This design decreases the distance between the FRET donor and the FRET acceptors on ConA to maximize the FRET efficiency upon binding of the ligand to ConA. This work specifically compares the FRET efficiency and sensitivity of this new competing ligand with a traditional dextran ligand, showing that the new ligand has improved characteristics. This work also tested the long-term thermal stability of the assay based on this new competing ligand and displayed a MARD of less than 10% across the physiological range of glucose after 30 days incubation at 37 °C. Ultimately, this new type of fluorescent ligand has the potential to significantly improve the accuracy of continuous glucose monitoring devices based on the competitive binding sensing approach.

Competitive binding assays utilizing concanavalin A (ConA) have the potential to be the basis of improved continuous glucose monitoring devices. However, the efficacy and lifetime of these assays have been limited, in part, by ConA's instability due to its thermal denaturation in the physiological environment (37 °C, pH 7.4, 0.15 M NaCl) and its electrostatic interaction with charged molecules or surfaces. These undesirable interactions change the constitution of the assay and the kinetics of its behavior over time, resulting in an unstable glucose response. In this work, poly(ethylene glycol) (PEG) chains are covalently attached to lysine groups on the surface of ConA (i.e., PEGylation) in an attempt to improve its stability in these environments. Dynamic light scattering measurements indicate that PEGylation significantly improved ConA's thermal stability at 37 °C, remaining stable for at least 30 days. Furthermore, after PEGylation, ConA's binding affinity to the fluorescent competing ligand previously designed for the assay was not significantly affected and remained at ~5.4 × 10(6) M(-1) even after incubation at 37 °C for 30 days. Moreover, PEGylated ConA maintained the ability to track glucose concentrations when implemented within a competitive binding assay system. Finally, PEGylation showed a reduction in electrostatic-induced aggregation of ConA with poly(allylamine), a positively charged polymer, by shielding ConA's charges. These results indicate that PEGylated ConA can overcome the instability issues from thermal denaturation and nonspecific electrostatic binding while maintaining the required sugar-binding characteristics. Therefore, the PEGylation of ConA can overcome major hurdles for ConA-based glucose sensing assays to be used for long-term continuous monitoring applications in vivo.

Researchers have found that decreased levels of circulating citrulline could be an indicator of intestinal failure. Typically, this amino acid, which is produced by the intestinal mucosa cells, circulates in the blood at a physiological level of ∼40 μM. The current methodology for measuring this level involves the use of bulky equipment, such as mass spectroscopy and analysis at a central laboratory, which can delay diagnosis. Therefore, the current detection method is unsuited for routine monitoring at a doctor’s office. Our research group proposes the development of a point-of-care (POC) device to overcome this issue. The proposed device utilizes surface-enhanced Raman spectroscopy (SERS) coupled with a specifically designed aptamer, capable of binding to citrulline, conjugated to colloidal gold nanoparticles. The assay is then embedded within a vertical flow paper-fluidic platform as a deliverable at the POC, and a handheld Raman spectrometer (638-nm excitation) was used to interrogate the sample. Results showed good dynamic range and specificity with an average 73% decrease in SERS signal intensity with increasing concentrations of citrulline (0 to 50 μM) in phosphate-buffered saline compared to its controls: glycine, glutamine, histidine, and valine, which showed less than 10% average decrease in the presence of 200 μM of each analyte. Further, the limit of detection (LOD) within a chip was determined to be 0.56 μM, whereas the LOD across chips was below 10 μM.

Human biomarkers are indicative of the body’s relative state prior to the onset of disease, and sometimes before symptoms present. While blood biomarker detection has achieved considerable success in laboratory settings, its clinical application is lagging and commercial point-of-care devices are rare. A physician’s ability to detect biomarkers such as microRNA-17, a potential epigenetic indicator of preeclampsia in pregnant woman, could enable early diagnosis and preventive intervention as early as the 1st trimester. One detection approach employing DNA-functionalized nanoparticles to detect microRNA-17, in conjunction with surface-enhanced Raman spectroscopy (SERS), has shown promise but is hindered, in part, by the use of large and expensive benchtop Raman microscopes. However, recent strides have been made in developing portable Raman systems for field applications. Characteristics of the SERS assay responsible for strengthening the assay’s plasmonic response were explored, whilst comparing the results from both benchtop and portable Raman systems. The Raman spectra and intensity of three different types of photoactive molecules were compared as potential Raman reporter molecules: chromophores, fluorophores, and highly polarizable small molecules. Furthermore, the plasmonic characteristics governing the formation of SERS colloidal nanoparticle assemblies in response to DNA/miRNA hybridization were investigated. There were significant variations in the SERS enhancement in response to microRNA-17 using our assay depending on the excitation lasers at wavelengths of 532 nm and 785 nm, depending on which of the three different Raman systems were used (benchtop, portable, and handheld), and depending on which of the three different Raman reporters (chromophore, fluorophore, or Raman active molecule) were used. Analysis of data obtained did indicate that signal enhancement was better for the chromophore (MGITC) and Raman active molecule (DTNB) than it was for the fluorophore (TRITC) and that, although it is possible to obtain enhancements when using excitation lasers that do not directly coincide with the optical properties of the Raman reporter molecule, clearly the enhancements are more significant when it reaches to the characteristic wavelengths of those molecules.

The controlled assembly of plasmonic nanoparticles by a molecular binding event has emerged as a simple yet sensitive methodology for protein detection. Metallic nanoparticles (NPs) coated with functionalized aptamers can be utilized as biosensors by monitoring changes in particle optical properties, such as the LSPR shift and enhancement of the SERS spectra, in the presence of a target protein. Herein we test this method using two modified aptamers selected for the protein biomarker interleukin 6, an indicator of the dengue fever virus and other diseases including certain types of cancers, diabetes, and even arthritis. IL6 works by inducing an immunological response within the body that can be either anti-inflammatory or pro-inflammatory. The results show that the average hydrodynamic diameter of the NPs as measured by Dynamic Light Scattering was ∼42 nm. After conjugation of the aptamers, the peak absorbance of the AgNPs shifted from 404 to 408 nm indicating a surface modification of the NPs due to the presence of the aptamer. Lastly, preliminary results were obtained showing an increase in SERS intensity occurs when the IL-6 protein was introduced to the conjugate solution but the assay will still need to be optimized in order for it to be able to monitor varying concentration changes within and across the desired range.

Competitive binding assays comprised of the protein Concanavalin A (ConA) have shown potential for use in continuous glucose monitoring devices. However, its time-dependent, thermal instability can impact the lifetime of these ConA based assays. In an attempt to design sensors with longer in vivo lifetimes, different groups have immobilized the protein to various surfaces. For example, Ballerstadt et al. have shown that immobilizing ConA onto the interior of a micro-dialysis membrane and allowing dextran to be freely suspended within solution allowed for successful in vivo glucose sensing up to 16 days. This work explores the glucose response of an assay comprised of modified ConA and a single fluorescently labeled competing ligand in free solution to increase the in vivo sensing lifetime without immobilization,. The behavior of this assay in the presence of varying glucose concentrations is monitored via fluorescence anisotropy over a 30 day period.

We present results from experiments in using several pitch representations for jazz-oriented musical tasks performed by a recurrent neural network. We have run experiments with several kinds of recurrent networks for this purpose, and have found that Long Short-term Memory networks provide the best results. We show that a new pitch representation called Circles of Thirds works as well as two other published representations for these tasks, yet it is more succinct and enables faster learning. We then discuss limited results using other types of networks on the same tasks.

DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies.

Basefreies Tris(trimethylsilyl)methyl-Lithium, Tsi–Li, reagiert mit den Trihalogeniden der Erdmetalle (MHal3 mit M = Al, Ga, In und Hal = Cl, Br, I) primar unter Bildung der Metallate [Tsi–MHal3]Li. Vor allem mit den schwereren Halogeniden von Ga und In findet aber mit uberschussigem Tsi–Li zusatzlich zur simplen Metathese gleichzeitig auch eine Methylierung statt, die in unterschiedlichem Mase die Mono- und Dimethylverbindungen Tsi–M(Me)Hal (M = Ga, In; Hal = I), Tsi–MMe2 (M = Ga) bzw. das Bis-(trisyl)derivat (Tsi)2InMe sowie stets als Nebenprodukt noch 1,3-Disilacyclobutan ergibt. Durch mehrmalige, fraktionierende Kristallisationen oder Sublimationen konnten Vertreter dieser Verbindungstypen aus den Reaktionsmischungen isoliert und mit spektroskopischen Methoden (1H, 13C, 29Si-NMR; IR, Raman) sowie zum Teil rontgenographisch charakterisiert werden. FeCl3 reagiert mit Tsi–Li (Verhaltnis 1 : 3) in Toluol bei 55–60 °C unter Reduktion, wobei rotviolettes Fe(Tsi)2, 1,1,1-Tris(trimethylsilyl)-2-phenylethan und geringe Mengen an Tsi–Cl gebildet werden. Fe(Tsi)2 ist monomer, kristallisiert in der monoklinen Raumgruppe C2/c und hat ein lineares C–Fe–C-Skelett mit Fe–C-Bindungsabstanden von 204,5(4) pm. The Variable Reaction Behaviour of Base-free Tris(trimethylsilyl)methyl Lithium with Trihalogenides of Earth-Metals and Iron Base-free tris(trimethylsilyl)methyl Lithium, Tsi–Li, reacts with the earth-metal trihalogenides (MHal3 with M = Al, Ga, In and Hal = Cl, Br, I) primarily to give the metallates [Tsi–MHal3]Li. Simultaneous to this simple metathesis a methylation also takes place, mainly with heavier halogenides of Ga and In with excess Tsi–Li, forming the mono and dimethyl compounds Tsi–M(Me)Hal (M = Ga, In; Hal = I), Tsi–MMe2 (M = Ga), and the bis(trisyl)derivative (Tsi)2InMe, respectively and the main by-product 1,3-disilacyclobutane. Representatives of each type of compound have been isolated by fractionating crystallizations or sublimations and characterized by spectroscopic methods (1H, 13C, 29Si NMR, IR, Raman) and X-ray elucidations. Reduction takes place, when FeCl3 reacts with Tsi–Li (1 : 3 ratio) in toluene at 55–60 °C, yielding red-violet Fe(Tsi)2, 1,1,1-tris(trimethylsilyl)-2-phenyl ethane and low amounts of Tsi–Cl. Fe(Tsi)2 is monomeric, crystallizes in the monoclinic space group C2/c and consists of a linear C–Fe–C skeleton with d(Fe–C) of 204,5(4) pm.

A new reagent, 1-(9-anthracenylmethyl)piperazine (MAP), was used for the derivatization of airborne 1,6-hexamethylene diisocyanate (HDI) and polyisocyanates generated during spray-painting operations. The new reagent, which offers enhanced sensitivity and uniformity of response to both the monomeric and oligomeric forms of HDI, was compared directly with 1-(2-methoxyphenyl)piperazine (MOP), the currently employed derivatizing reagent used in the National Institute for Occupational Safety and Health Method 5521. Both the validity of the side-by-side sampling protocol and the efficacy of two derivatizing reagents were evaluated in field studies. The analytical results indicate that there is no significant difference at the 95% confidence level in the concentration of polyisocyanate in the aerosol as determined by two impingers containing MAP and a third containing MOP when these are positioned in a side-by-side-by-side arrangement.

We describe 25 cases of Wegener's granulomatosis presenting with solitary lung lesions to compare the clinical and pathologic findings in these cases with those of the more common multifocal disease and also to evaluate the clinical significance of solitary lung lesions occurring in the absence of extrapulmonary disease. The clinical findings in our patients with solitary Wegener's were similar to those in generalized disease. Men were affected slightly more often than women, and the average age of onset was 53 years. Likewise, no major pathologic differences were found between solitary and multifocal disease. Classic necrotizing granulomatous inflammation and necrotizing vasculitis were the most common findings, although other variants were occasionally encountered, including the eosinophilic variant (two cases), the bronchocentric variant (one case), and small-vessel vasculitis and capillaritis (one case). Three cases had prominent areas of bronchiolitis obliterans-organizing pneumonia. Progressive disease occurred in all seven patients who manifested solitary lung lesions without extrapulmonary involvement and were not treated because the diagnosis was initially unrecognized. Pathologists need to be alert to the possibility of Wegener's granulomatosis causing solitary lung lesions because treatment should be promptly instituted.

Bei der Reaktion zwischen elementarem Selen und MBH4 (1 : 1) (M = Na, Li) in Triglyme (Diglyme) entsteht unter Wasserstoffabspaltung M2[H3BSeSeBH3] 1. Bei der Behandlung von 1 mit uberschussigem B2H6 oder THF · BH3 wird die SeSe-Bindung gespalten und es entsteht unter erneuter Wasserstoffabspaltung M[H3B-μ2-Se(B2H5)] 2. Aus Na · 2 und [(C6H5)4P]Br entsteht [(C6H5)4P] · 2, welches tetragonal in der Raumgruppe I4 (Nr. 82) kristallisiert. Kation und Anion sind im Kristall fehlgeordnet. Strukturinformationen konnten aus den 11B-, 77Se- und 1J(11B1H)-Daten einerseits und den IR- und Raman-spektroskopischen Untersuchungen andererseits erhalten werden. Aus SCF-Rechnungen erhielten wir die Strukturparameter fur das Anion 2. 2 kann sowohl als Addukt von Se an B3H8− als auch als bruckensubstituiertes Selena-Derivat des B2H6 aufgefast werden. Synthesis and Vibrational Spectroscopic Investigation of [H3BSeSeBH3]2− and [H3B-μ2-Se(B2H5)]− Crystal Structure and Theoretical Investigation of the Molecular Structure of [H3B-μ2-Se(B2H5)]− M2[H3BSeSeBH3] 1 is produced by the reaction between elemental selenium and MBH4 (1 : 1) in triglyme (diglyme), under dehydrogenation. 1 reacts with an excess of B2H6 to give M[H3B-μ2-Se(B2H5)] 2 which is also formed in the reaction of THF · BH3 with 1. These reactions proceed under cleavage of the SeSe bond and hydrogen evolution. [(C6H5)4]Br reacts with Na · 2 to form [(C6H5)4P] · 2 which crystallizes in the tetragonal space group I4 (Nr. 82). An X-ray structure determination failed because of disordering of the cation and anion. 11B, 77Se NMR shifts and 1J(11B1H) coupling constants as well as IR- and Raman spectroscopic investigations convey further structural information. Structural data of 2 have been calculated by SCF methods. The anion of 2 may be viewed either as an adduct of Se with B3H8−, or as a bridge substituted selena derivative of B2H6.

Actin stress fibers (SFs), bundles of actin filaments crosslinked by α-actinin and myosin II in non-muscle cells, are mechanosensitive structural elements that respond to applied stress and strain to regulate cell morphology, signal transduction and cell function. Results from various studies indicate that myosin-generated contraction extends SFs beyond their unloaded lengths and cells maintain fiber strain at an optimal level that depends on actomyosin activity (Lu et al., 2008). Stretching the matrix upon which cells adhere perturbs the cell-matrix traction forces and cells respond by actively re-establishing the preexisting level of force (Brown et al., 1998; Gavara et al., 2008). We have developed a sarcomeric model of SF networks (Kaunas et al., 2011) to predict the effects of stretch on SF reorganization depending on the rates of matrix stretching, SF turnover, and SF stress relaxation.Copyright © 2011 by ASME

Cytoskeletal tension enables cells to adhere, spread, contract, and migrate. In adherent, non-muscle cells such as endothelial cells and fibroblasts, tension is generated by actomyosin stress fibers applying traction forces to cell-matrix adhesions. Tension extends stress fibers beyond their unloaded lengths and cells maintain a preferred level of fiber strain that depends on actomyosin activity (Lu 2008). Stretching the matrix upon which cells adhere perturbs the cell-matrix traction forces and cells respond by actively re-establishing the pre-existing level of force (Gavara 2006). Sudden large increases or decreases in strain result in rapid stress fiber disassembly and reassembly (Lu 2008), suggesting that perturbing fiber strain from a preferred level stimulates stress fiber turnover.

Background Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency. Principal Findings Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts. Conclusions These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.

We have epitaxially grown various semiconductor hosts with Tm as rare earth element dopant to evaluate the excitation and decay mechanisms of its inner-atomic transitions in semiconductors. Electrical measurements revealed no characteristic influence of the Tm doping, besides a slight decrease of the carrier mobilities. We have detected Tm 3+ related emissions at 1.9 and 1.2 μm wavelenght. Highest intensities have been observed in GaInP:Tm samples. The spectra are similar in all semiconductor hosts under investigation. We have found indication that Tm is not only incorporated in regular lattice sites, but probably in the form of complexes. Further, we have done co-doping experiments with S (for n-type doping) and Zn (p-type doping) in combination with Tm. We have found only a slight influence of the doping partner on the optical properties, but no indication for optical transitions of Tm 2+ .

(Me2InN2C3H3)2 (Me CH3) crystallizes in the monoclinic space group P21/c with the lattice constants a 1727.0(2), b 2169.7(3), c 815.4(1) pm, β 92.64(1)° and Z = 8 (dimers). The unit cell contains two crystallographically independent molecules A and B which do not differ significantly. The In(-NN-)2 In skeleton of both dimeric molecules has a boat conformation; the structure has been refined to an R-value of 0.032.

The National Data Buoy Center (NDBC) maintains an extensive array of moored buoys around the world. Hence, mounting Acoustic Doppler Current Profilers (ADCPs) to these buoys has proven to be an avenue worth exploring. In a previous study done by Seim and Edwards [1], a downward-looking ADCP from NDBC buoy 41008 was compared to an upward-looking ADCP from the University of North Carolina at Chapel Hill (UNC) located in close proximity to test the validity of ADCP measurements made by a buoy-mounted ADCP. Since configurations of the systems were not standard, the two did not agree well. Since this time, NDBC has made several changes to the configuration of their ADCPs. This study is to again compare a NDBC downward-looking ADCP mounted to buoy 41036 to an upward-looking ADCP from the University of North Carolina at Wilmington (UNCW) mounted on the seafloor to test the reliability of NDBCs present buoy-mounted ADCP configuration. Both of these systems are located on the shallow continental shelf of Onslow Bay, North Carolina. An 18-day time series was obtained from each ADCP. Preliminary results show good agreement between the two systems. In light of the fact that the buoy-mounted system is subjected to movement by atmospheric and oceanic processes, further data conditioning is investigated to see if more precise environmental thresholds, specifically wave height thresholds, can be put in place for more accurate current measurements.

An evaluation of eight compounds for use as an internal standard in fire debris analysis was conducted. Tests were conducted on tetrachloroethylene, chlorobenzene, n-octylbenzene, 3-phenyltolune, and deuterated compounds toluene-d8, styrene-d8, naphthalene-d8, and diphenyl-d10 to measure the extraction efficiency of each compound in the presence of an interfering volatile compound (carbon disulfide). Other tests were conducted to evaluate whether or not the presence of an ignitable liquid or pyrolysis/combustion products from fire debris would interfere with the identification of these compounds when used as an internal standard. The results showed that while any of the eight compounds could be used as an internal standard in fire debris analysis, the more volatile compounds (toluene-d8, tetrachloroethylene, chlorobenzene, and styrene-d8) showed better extraction efficiencies at room temperature than when heated to 60 degrees C. Each of the less volatile compounds (naphthalene-d8, diphenyl-d10, n-octylbenzene, and 3-phenyltolune) performed well during extraction at 60 degrees C, while naphthalene-d8 showed better extraction efficiency in the presence of competing volatiles when extracted at room temperature. Language: en