Search

Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cutoff (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP with low molecular weight (11 kDa and 17 kDa) at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

Extracellular vesicles (EVs) are increasingly being recognized as important vehicles for intercellular communication and as promising sources for biomarker discovery. Because the state of protein post-translational modifications (PTMs) such as phosphorylation and glycosylation can be a key determinant of cellular physiology, comprehensive characterization of protein PTMs in EVs can be particularly valuable for early-stage diagnostics and monitoring of disease status. However, the analysis of PTMs in EVs has been complicated by limited amounts of purified EVs, low-abundance PTM proteins, and interference from proteins and metabolites in biofluids. Recently, we developed an approach to isolate phosphoproteins and glycoproteins in EVs from small volumes of human plasma that enabled us to identify nearly 10,000 unique phosphopeptides and 1,500 unique N-glycopeptides. The approach demonstrated the feasibility of using these data to identify potential markers to differentiate disease from healthy states. Here we present an updated workflow to sequentially isolate phosphopeptides and N-glycopeptides, enabling multiple PTM analyses of the same clinical samples. In this updated workflow, we have improved the reproducibility and efficiency of EV isolation, protein extraction, and phosphopeptide/N-glycopeptide enrichment to achieve sensitive analyses of low-abundance PTMs in EVs isolated from 1 mL of plasma. The modularity of the workflow also allows for the characterization of phospho- or glycopeptides only and enables additional analysis of total proteomes and other PTMs of interest. After blood collection, the protocol takes 2 d, including EV isolation, PTM/peptide enrichment, mass spectrometry analysis, and data quantification.

NbRabF1, a small GTPase from Nicotiana benthamiana and a homolog of Arabidopsis thaliana Ara6, plays a key role in regulating Bamboo mosaic virus (BaMV) movement by vesicle transport between endosomal membranes. Reducing the expression of NbRabF1 in N. benthamiana by virus-induced gene silencing decreased the accumulation of BaMV, and with smaller infection foci on inoculated leaves, but had no effect in protoplasts. Furthermore, transient expression of NbRabF1 increased the accumulation of BaMV in inoculated leaves. Thus, NbRabF1 may be involved in the cell-to-cell movement of BaMV. The potential acyl modification sites at the second and third amino acid positions of NbRabF1 were crucial for membrane targeting and BaMV accumulation. The localization of mutant forms of NbRabF1 with the GDP-bound (donor site) and GTP-bound (acceptor site) suggested that NbRabF1 might regulate vesicle trafficking between the Golgi apparatus and plasma membrane. Furthermore, GTPase activity could also be involved in BaMV cell-to-cell movement. Overall, in this study, we identified a small GTPase, NbRabF1, from N. benthamiana that interacts with its activation protein NbRabGAP1 and regulates vesicle transport from the Golgi apparatus to the plasma membrane. We suggest that the BaMV movement complex might move from cell to cell through this vesicle trafficking route.

Plasma membrane-associated cation-binding protein 1-like protein of Nicotiana benthamiana plays a role in defense against Bamboo mosaic virus by binding the viral replicase and restricting viral intercellular movement., To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants. Further analysis indicated no significant difference in the accumulation of BaMV in NbPCaP1L knockdown and control protoplasts, suggesting NbPCaP1L may affect cell-to-cell movement of BaMV. Using a viral vector expressing green fluorescent protein in the knockdown plants, the mean area of viral focus, as determined by fluorescence, was found to be larger in NbPCaP1L knockdown plants. Orange fluorescence protein (OFP)-fused NbPCaP1L, NbPCaP1L-OFP, was expressed in N. benthamiana and reduced the accumulation of BaMV to 46%. To reveal the possible interaction of viral protein with NbPCaP1L, we performed yeast two-hybrid and co-immunoprecipitation experiments. The results indicated that NbPCaP1L interacted with BaMV replicase. The results also suggested that NbPCaP1L could trap the BaMV movement RNP complex via interaction with the viral replicase in the complex and so restricted viral cell-to-cell movement.

Salmonella is one of the most common pathogens associated with foodborne illness in chickens. Food outbreaks from this pathogen haven’t declined in the past 15 years according to the data from Centers for Disease Control and Prevention. It is our goal to improve food safety monitoring in this area by developing a real time Salmonella detection sensor on food surfaces. Previously, we demonstrated the use of phage C4-22 immobilized onto a rapid magnetoelastic (ME) biosensor for use as a front-line detection ligand to detect all Salmonella enterica serotypes in Tris Buffer Saline (TBS). In this study, by using fluorescent imaging, the phage peptide binding to Salmonella enterica serotype Typhimurium cells is again confirmed. Moreover, we constructed two detection models to evaluate the detection of Salmonella on/in chicken meat using the phage coated ME sensors. In the chicken surface detection method, phage C4-22 sensors demonstrated more than 12 times higher Salmonella binding capacity than the control sensors with no phage for the Salmonella spiked at the concentration of 7.86 × 105 cfu/mm2. In the second model, phage sensors were placed at different depths inside the chicken breast (0.1 cm; 0.5 cm; 1.0 cm below the meat surface) after surface inoculation of Salmonella. The second detection system showed that 23.27%–33% of the inoculated Salmonella cells absorbed inside the chicken breast fillets below 0.1 cm of the surface. The data for direct detection on chicken showed that phage C4-22 ME biosensors bind ultimately when there are high concentrations of Salmonella on the chicken surface. The results also suggest that the phage sensors can detect Salmonella effectively when the bacterial contaminants are absorbed into the chicken, and are not detectable by the surface detection method.

The prevalence of upper tract urothelial carcinoma (UTUC) in Taiwan is relatively higher than thatin Western countries. Aristolochic acid (AA), which is widely used in traditional Chinese herbology, is now recognized to be one of the carcinogens for UTUC. Numerous UTUC patients have chronic kidney diseases or end-stage renal diseases, however, little literature hasreported on theoncogenic pathway of AA-related UTUC. The aim of our study was to identify the potential target treatment for AA-related UTUC. Here, we established an AA pre-exposure followed bya 3-methylcholanthrene (MCA) stimulus tumorigenic cell model. We not only demonstrated that AA pre-exposure MCA stimulus tumorigenic cells have more behaviors of cell migration and invasion by enhancing the metalloproteinases (MMP) activity, which is compatible with clinical findings of AA-related UTUC, but we also validated that AA pre-exposure MCA stimulus tumorigeniccells could be activated through the mitogen-activated protein kinases (MAPK) pathway. We further dissected the route of the MAPK pathway and found that the p38 and extracellular signal regulated kinases (ERK) sub-pathways might play essential roles in AA pre-exposure urothelial cancer cell lines. This consequence was also corroborated with a tissue study in AA-exposed patients.

A helium (He) atmospheric-pressure dielectric barrier discharge jet (DBDjet) is used to pattern 1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane (PFDTS)-coated cellulose paper with a shadow mask. DBDjet processing under appropriate conditions can be used to form hydrophilic patterns. The properties of the DBDjet-patterned PFDTS-coated paper are investigated through water contact angle measurement, scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS), and the results indicate the successful deposition and removal of PFDTS before/after DBDjet treatment. The successful routing and mixing of liquid in the hydrophilic stripes are demonstrated, and a glucose concentration colorimetric test is demonstrated using this DBDjet-patterned PFDTS-coated paper.

Host cell proteins (HCPs) impurities are critical quality attributes that have the potential to negatively impact the quality and safety profile of a biopharmaceutical product. Since HCPs often are present at low levels, developing highly sensitive analytical method for their identification and quantitation is critical for process optimization and improvement to reduce them in the final drug product. While an enzyme-linked immunosorbent assay (ELISA) can capture and quantify overall HCP levels, liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool to monitor individual HCP levels during the purification process development. The massive dynamic range of protein species present in a therapeutic antibody is a major challenge for mass spectrometry-based methods to detect low-abundance HCP impurities. This study reports a powerful strategy to identify HCPs in antibody drug substance by applying ProteoMiner enrichment with optimized conditions followed by shotgun proteomic analysis. Using this strategy, we observed that the low abundance HCPs were enriched up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP at a concentration as low as 0.05 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 500 HCPs were confidently identified, which tripled the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were enriched between 100 and 400-fold, highlighting that our method enriches and equalizes all proteins thus improving the sensitivity of HCP identification and quantification.

This paper investigates the accuracy of surface-scanning measurement of a wireless magnetoelastic (ME) biosensor for direct pathogen detection on solid surfaces. The model experiments were conducted on the surface of a at polyethylene (PE) plate. An ME biosensor (1 mm x 0.2 mm x 30 µm) was placed on the PE surface, and a surface-scanning detector was aligned to the sensor for wireless resonant frequency measurement. The position of the detector was accurately controlled by using a motorized three-axis translation system (i.e., controlled X, Y, and Z positions). The results showed that the resonant frequency variations of the sensor were -125 to +150 Hz for X and Y detector displacements of ± 600 µm and Z displacements of +100 to +500 µm. These resonant frequency variations were small compared to the sensor's initial resonant frequency (˂ 0.007% of 2.2 MHz initial resonant frequency) measured at the detector home position, indicating high accuracy of the measurement. In addition, the signal amplitude was, as anticipated, found to decrease exponentially with increasing detection distance (i.e., Z distance). Finally, additional experiments were conducted on the surface of cucumbers. Similar results were obtained.

Glycoproteins comprise more than half of current FDA-approved protein cancer markers, but the development of new glycoproteins as disease biomarkers has been stagnant. Here we present a pipeline to develop glycoproteins from extracellular vesicles (EVs) through integrating quantitative glycoproteomics with a novel reverse phase glycoprotein array and then apply it to identify novel biomarkers for breast cancer. EV glycoproteomics show promise in circumventing the problems plaguing current serum/plasma glycoproteomics and allowed us to identify hundreds of glycoproteins that have not been identified in blood. We identified 1,453 unique glycopeptides representing 556 glycoproteins in EVs, among which 20 were verified significantly higher in individual breast cancer patients. We further applied a novel glyco-specific reverse phase protein array to quantify a subset of the candidates. Together, this study demonstrates the great potential of this integrated pipeline for biomarker discovery.

This paper investigates the effects of surface-scanning detector position on the resonant frequency and signal amplitude of a wireless magnetoelastic (ME) biosensor for direct pathogen detection on solid surfaces. The experiments were conducted on the surface of a flat polyethylene (PE) plate as a model study. An ME biosensor (1 mm × 0.2 mm × 30 μm) was placed on the PE surface, and a surface-scanning detector was brought close and aligned to the sensor for wireless resonant frequency measurement. The position of the detector was accurately controlled by using a motorized three-axis translation system (i.e., controlled X, Y, and Z positions). The results showed that the resonant frequency variations of the sensor were -125 to +150 Hz for X and Y detector displacements of ±600 μm and Z displacements of +100 to +500 μm. These resonant frequency variations were small compared to the sensor's initial resonant frequency (< 0.007% of 2.2 MHz initial resonant frequency) measured at the detector home position, indicating high accuracy of the measurement. In addition, the signal amplitude was, as anticipated, found to decrease exponentially with increasing detection distance (i.e., Z distance). Finally, additional experiments were conducted on the surface of cucumbers. Similar results were obtained.

This paper demonstrates a highly sensitive surface-scanning detector used for magnetoelastic (ME) biosensors for the detection of Salmonella on the surface of a polyethylene (PE) food preparation surface. The design and fabrication methods of the new planar spiral coil are introduced. Different concentrations of Salmonella were measured on the surface of a PE board. The efficacy of Salmonella capture and detection is discussed.

Phage Display technology provides a mechanism for us to make bio-recognition elements on biosensors for detection of Salmonella enterica serovars. In the procedure, the filamentous M13 bacteriophage is used for acquiring peptides that have a high affinity for the target recognition. Our approach in this study was to develop peptide structures in the pIII region of this thread-shaped virus. A phage pIII library was used to perform biopanning for the phage clones to bind the target Salmonella serovars. The clones were bound, washed, eluted and amplified four times. Then, the phage peptides were sequenced tested for specificity using ELISA procedures. In this project to make a biosensor for all relevant Salmonella enterica serovars, we used common LPS salmonellae antigens as targets in the biopanning procedure. This enabled us to have a phage probe specific for all serovars of Salmonella enterica excluding the typhoid organisms. The final phage was then immobilized onto an electromagnetic platform to complete the biosensor, which gives us the real-time ability to measure resonance changes that indicate mass loading. The mass loading is an indication of binding to the target cells. Our current data with an ELISA procedure show the phage probe’s high affinity for salmonellae, very low cross-reactivity with Escherichia coli, Shigella, and no cross-reactivity to Staphylococcus aureus and Listeria monocytogenes. The biosensor with the phage showed that the capture ability for Salmonella serovars is thirty times higher than the control sensor. This biosensor is a candidate for detection of Salmonella in food and other settings.

Efforts were made to incorporate the phage Magnetoelastic (ME) biosensor in FDA's Spinach Soaking procedures according to FDA 2015 BAM method. Three soaking materials (Lactose broth, LB broth, and Peptone water) and various soaking times were investigated. Using merely 100 Salmonella cells spiked on the produce surfaces, the phage biosensors detected Salmonella within 5 hours when soaking tomatoes in LB broth as opposed to taking up to 24 hours. Salmonella was detected on spinach leaves within 7 hours. These phage ME biosensors provide a promising rapid detection platform using LB broth in FDA's soaking procedures while shortening the incubation period.

One of the important applications for which phage-immobilized magnetoelastic (ME) biosensors are being developed is the wireless, on-site detection of pathogenic bacteria for food safety and bio-security. Until now, such biosensors have been constructed by immobilizing a landscape phage probe on gold-coated ME resonators via physical adsorption. Although the physical adsorption method is simple, the immobilization stability and surface coverage of phage probes on differently functionalized sensor surfaces need to be evaluated as a potential way to enhance the detection capabilities of the biosensors. As a model study, a filamentous fd-tet phage that specifically binds streptavidin was adsorbed on either bare or surface-functionalized gold-coated ME resonators. The surface functionalization was performed through the formation of three self-assembled monolayers with a different terminator, based on the sulfur-gold chemistry: AC (activated carboxy-terminated), ALD (aldehyde-terminated), and MT (methyl-terminated). The results, obtained by atomic force microscopy, showed that surface functionalization has a large effect on the surface phage coverage (46.8%, 49.4%, 4.2%, and 5.2% for bare, AC-, ALD-, and MT-functionalized resonators, respectively). In addition, a direct correlation of the observed surface phage coverage with the quantity of subsequently captured streptavidin-coated microbeads was found by scanning electron microscopy and by resonance frequency measurements of the biosensors. The differences in surface phage coverage on the differently functionalized surfaces may then be used to pattern the phage probe layer onto desired parts of the sensor surface to enhance the detection capabilities of ME biosensors.

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.

This paper presents the results of a study that investigates the optimization of phage chemistry during the fabrication of magnetoelastic (ME) biosensors for the detection of Salmonella typhimurium or Bacillus anthracis spores. The bundling characteristics of the phage filaments limit the ability of the biosensor to bind bacterial cells/spores. Experiments were performed to determine the proper phage concentration for the prevention of bundling in aqueous environments. Based on the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) results, which verify the structure of phage under different concentrations and binding numbers of target species to the sensor surface, we found that phage concentrations of 1011 vir/ml exhibit the best sensor performance in terms of binding sensitivity. Additionally, the sensors immobilized with phage under this condition were tested in a flowing liquid system using S. typhimurium and B. anthracis spores suspensions in concentrations ranging from 5 times101 to 5 times 108 cfu/ml, separately. As cells/spores are bound to a ME biosensor surface, the additional mass of the spores causes a decrease in the resonance frequency of the sensor. The frequency response curves of the ME biosensors as a function of exposure time were then measured, and the detection limit of the ME biosensor was determined to be 5 times 103 cfu/ml.

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1 mg/ml BSA (blocking agent) onto the magnetoelastic resonator’s surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor’s resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.

A series of one-dimensional (1D) coordination polymers, {[Cu(en)2][M(C5O5)2(H2O)m]·xH2O}n, (M = Mn (1), m = 4, x = 2; M = Co (2), Ni (3), Cu (4), Zn (5), m = 2, x = 0; en = ethylenediamine; C5O52− = croconate, dianion of 4,5-dihydroxycyclopent-4-ene-1,2,3-trione), using [M(C5O5)2(H2O)n]2− dianion as a building block has been synthesized and characterized by the X-ray diffraction method. Structural determination reveals that, in compounds 1−5, one [M(C5O5)2(H2O)n]2− dianion coordinates via two oxygen (croconate) donors to the axial sites of two Cu(II) centers of [Cu(en)2]2+ cations to form one-dimensional (1D) linear (1, 3 and 4) or zigzag (2 and 5) chains of alternating [M(C5O5)2(H2O)n]2− and [Cu(en)2]2+ units. The croconate acts as a bridging ligand with three coordination modes as μ1,3-bismonodentate for 1, μ1,2,3-bidentate/monodentate for 2 and 5, and μ1,2,4-bidentate/monodentate for 3 and 4, respectively. In addition, a byproduct, [Cu(en)(C5O5)(H2O)]2 (6), which is cocrystallized with 4, shows a dimer...

A biosensor for the detection of biological warfare agents (Bacillus anthracis spores) was developed that combines the phage display technique with a magnetoelastic wireless detection platform. The affinity-based biosensor utilizes a phage-derived diagnostic probe as the biomolecular recognition element to capture target agents multivalently. Upon binding of the target agent to the sensor surface, the resonance frequency of the magnetoelastic biosensors decreases due to the additional mass of the target agent. Scanning electron microscopy was used to confirm binding of spores to the sensor surface. The sensitivity of the magnetoelastic acoustic sensor was tested to be 130 Hz per order of magnitude of spore concentration with a detection limit of 103 spores/ml. The specificity of the sensors was tested against spores of other closely related Bacillus species and a large preferential binding to Bacillus anthracis spores was observed. The longevity of the phage based biosensor was compared to traditional antibody based biosensors and found to exhibit a much longer life

Real-time in-situ detection of pathogenic bacteria on fresh food surfaces was accomplished with phage-based magnetoelastic (ME) biosensors. The ME biosensor is constructed of a small rectangular strip of ME material that is coated with a biomolecular recognition element (phage, antibodies or proteins, etc.) that is specific to the target pathogen. This mass-sensitive ME biosensor is wirelessly actuated into mechanical resonance by an externally applied time-varying magnetic field. When the biosensor binds with target bacteria, the mass of the sensor increases, resulting in a decrease in the sensor's resonant frequency. In order to compensate for nonspecific binding, control biosensors without phage were used in this experiment. In previous research, the biosensors were measured one by one. However, the simultaneous measurement of multiple sensors was accomplished in this research, and promises to greatly shorten the analysis time for bacterial detection. Additionally, the use of multiple biosensors enables the possibility of simultaneous detection of different pathogenic bacteria. This paper presents results of experiments in which multiple phage-based ME biosensors were simultaneously monitored. The E2 phage and JRB7 phage from a landscape phage library served as the bio-recognition element that have the capability of binding specifically with Salmonella typhimurium and B. anthracis spores, respectively. Real-time in-situ detection of Salmonella typhimurium and B. anthracis spores on food surfaces are presented.

Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis -acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

Phage-based magnetoelastic (ME) biosensors have been studied as an in-situ, real-time, wireless, direct detection method of foodborne pathogens in recent years. This paper investigates an ME biosensor method for the detection of Salmonella Typhimurium on fresh spinach leaves. A procedure to obtain a concentrated suspension of Salmonella from contaminated spinach leaves is described that is based on methods outlined in the U.S. FDA Bacteriological Analytical Manual for the detection of Salmonella on leafy green vegetables. The effects of an alternative pre-enrichment broth (LB broth vs. lactose broth), incubation time on the detection performance and negative control were investigated. In addition, different blocking agents (BSA, Casein, and Superblock) were evaluated to minimize the effect of nonspecific binding. None of the blocking agents was found to be superior to the others, or even better than none. Unblocked ME biosensors were placed directly in a concentrated suspension and allowed to bind with Salmonella cells for 30 min before measuring the resonant frequency using a surface-scanning coil detector. It was found that 7 h incubation at 37 °C in LB broth was necessary to detect an initial spike of 100 cfu/25 g S. Typhimurium on spinach leaves with a confidence level of difference greater than 95% (p < 0.05). Thus, the ME biosensor method, on both partly and fully detection, was demonstrated to be a robust and competitive method for foodborne pathogens on fresh products.

In this paper the performance of a magnetoelastic biosensor detection system for the simultaneous identification of B. anthracis spores and S. typhimurium was investigated. This system was also designed for selective in-situ detection of B. anthracis spores in the presence a mixed microbial population. The system was composed of a reference sensor (devoid of phage), an E2 phage sensor (coated with phage specific to S. typhimurium) and a JRB7 phage sensor (coated with phage specific to B. anthracis spores). When cells/spores are bound to the specific phage-based ME biosensor surface, only the resonance frequency of the specific sensor changed. The instantaneous response of the multiple sensor system was studied by exposing the system to B. anthracis spores and S. typhimurium suspensions sequentially. A detection limit of 1.6×103 cfu/mL and 1.1×103 cfu/m was observed for JRB7 phage sensor and E2 phage sensor, respectively. Additionally, the performance of the system was also evaluated by exposure to a flowing mixture of B. anthracis spores (5×101-5×108 cfu/ml) in the presence of B. cereus spores (5×107 cfu/ml). Only the JRB7 phage biosensor responded to the B. anthracis spores. Moreover, there was no appreciable frequency change due to non-specific binding when other microorganisms (spores) were in the mixture. A detection limit of 3×102 cfu/mL was observed for JRB7 phage sensor. The results show that the multi-sensor detection system offers good performance, including good sensitivity, selectivity and rapid detection.

This paper presents the results of a study that investigates the effect of salt concentration on the immobilization of phages during the fabrication of magnetoelastic (ME) biosensors for the detection of Bacillus anthracis spores. The concentration of salt in the solution used to deposit and immobilize the phage is found to alter phage bundling characteristics and, hence, influence the ability of the biosensor to bind spores. ME biosensors were fabricated using different salt concentrations in the phage solution, and were then exposed to solutions containing known amounts of B. anthracis spores. The frequency response curves of the ME biosensors, were then measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of B. anthracis spores to the phage-coated ME biosensors. Transmission electron microscopy (TEM) was used to verify the structure of phage under different salt concentrations. Results showed that moderate concentrations of salt (420 mM NaCl) in the phage solution results in an optimal distribution of immobilized phages on the sensor surface and, hence, better binding of spores to the biosensor.

Increased renal ANP synthesis, but decreased or unchanged cardiac ANP synthesis in water-deprived and salt-restricted rats. Background Experiments were performed to examine the effect of water deprivation and salt restriction on ANP synthesis in the kidneys and hearts of normal rats. Methods A 4-day water deprivation (WD) and 7-day salt restriction (SR; 0.01% NaCl) were performed in 12 and 14 rats, respectively. Atrial natriuretic peptide (ANP) mRNA expression in the kidney was assessed with reverse transcription-polymerase chain reaction coupled with Southern blot hybridization, while the ANP mRNA in the hearts was measured by Northern blot hybridization. ANP and angiotensin II concentrations in the extracted plasma were measured by radioimmunoassay. The molecular form of renal ANP-like protein was characterized by reverse phase—high-performance liquid chromatography (RP-HPLC). Results Renal outer and inner medullary ANP mRNA showed a respective 11-fold and ninefold increase in WD rats, and an eightfold and fivefold increase in SR rats as compared to corresponding control groups. Inversely, cardiac atrial ANP mRNA and plasma ANP were decreased in WD rats, whereas they did not change in the SR group. Plasma angiotensin II concentration increased in conjunction with the decrease of urine sodium excretion in both groups. RP-HPLC analysis revealed a 45% extraction of ANP in the WD rat kidneys, whereas only 3% ANP in the control kidneys migrated in a molecular form similar to cardiac atrial proANP. Conclusions Our results demonstrate that water deprivation and salt restriction markedly enhance renal ANP mRNA, whereas water deprivation suppresses cardiac atrial ANP mRNA and plasma ANP concentrations. The current study indicates that renal ANP and cardiac atrial ANP appear to be two distinct systems regulated by different mechanisms and possibly exhibiting different intra-renal paracrine and systemic endocrine functions.