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This article presents an investigation of the effect of salt and phage concentrations on the binding affinity of magnetoelastic (ME) biosensors. The sensors were fabricated by immobilizing filamentous phage on the ME platform surface for the detection of Bacillus anthracis spores. In response to the binding of spores to the phage on the ME biosensor, a corresponding decrease occurs in resonance frequency. Transmission electron microscopy (TEM) was used to verify the structure of phage under different combinations of salt/phage concentration. The chemistry of the phage solution alters phage bundling characteristics and, hence, influences both the sensitivity and detection limit of the ME biosensors. The frequency responses of the sensors were 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 spores to the sensor surface. This showed that 420 mM salt at a phage concentration of 1 x 10(11) vir/mL results in an optimal distribution of immobilized phages on the sensor surface, consequently promoting better binding of spores to the biosensor's surface. Additionally, the sensors immobilized with phage under this condition were exposed to B. anthracis spores in different concentrations ranging from 5 x 10(1) to 5 x 10(8) cfu/mL in a flowing system. The results showed that the sensitivity of this ME biosensor was 202 Hz/decade.

A micro-scale, free standing, wireless biosensor has been developed using magnetoelastic particles composed of an amorphous iron–boron binary alloy. Upon the application of an external magnetic field, these particles exhibit a characteristic resonance frequency, determined by their size and mass, due to the phenomena of magnetoelasticity. The particles are produced using the microelectronic fabrication techniques of photolithography and physical vapor deposition (sputtering). The biosensor is formed by coating the magnetoelastic particle with a thin layer of gold and immobilizing a biomolecular recognition element (bacteriophage) on the surfaces. Bacteriophage genetically engineered to bind Bacillus anthracis spores was used in this set of experiments as the detection probe. Once these targeted spores come into contact with the biosensor, the phage will bind selectively with only that pathogen, thereby increasing the particle's mass and causing a shift in the resonance frequency. Due to the magnetic nature of the sensing platform, this resonance frequency shift may be detected remotely by a wireless scanning device, presenting a distinct advantage over other techniques. A good correlation between the actual number of spores bound to the sensors and the calculated attached mass, based upon resonance frequency shifts, was obtained from the experiments.

The magnetostrictive microcantilever (MSMC) as a high-performance transducer was introduced for the development of biosensors. The principle and characterization of MSMC are presented. The MSMC is wireless and can be easily actuated and sensed using magnetic field/signal. More importantly, the MSMC exhibits a high Q value and works well in liquid. The resonance behavior of MSMC is characterized in air at different pressures and in different liquids, respectively. It is found that the Q value of the MSMC in water reaches about 40. Although the density and viscosity of the surrounding media affect the resonance frequency and the Q value of MSMC, the density has a stronger influence on the resonance frequency and the viscosity has a stronger influence on the Q value, which result in that, for MSMC in air at pressure of less than 100 Pa, the resonance frequency of MSMC is almost independent of the pressure, while the Q value increases with decreasing pressure. MSMC array was developed and characterized. It is experimentally demonstrated that the characterization of an MSMC array is as simple as the characterization of a single MSMC. A filamentous phage against Salmonella typhimurium was utilized as bio-recognition unit to develop an MSMC based biosensor. The detection of S. typhimurium in water demonstrated that the MSMC works well in liquid.

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

Filamentous phage affinity-selected for streptavidin and Salmonella typhimurium from phage display libraries were transformed with chloroform into spherical forms then deposited as phage coat protein monolayers to quartz crystal microbalances (QCM) by Langmuir-Blodgett (LB) to prepare bioselective sensors. Maximum yield of spheroids was achieved by mixing 8.3 x 10-11 to 2.5 x 10-12 filamentous phage virions/ml with 1 ml chloroform for 60 seconds. Spheroid conversion and binding to S. typhimurium was confirmed by transmission electron microscopy. Phage coat monolayers incorporating phospholipid possessed high elasticity and transference to QCM substrates comparable to antibodies. Sensor responses to increasing concentrations of target analytes were characterized by rapid reaction, steady-state equilibrium, high sensitivity, and linear dose-response that followed mass theory for piezoelectric transducers. Scanning electron microscopy confirmed binding of target analytes to biosensors compared to controls. Phage-based sensors may allow detection of bacterial agents such as S. typhimurium in food safety and security applications.

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 with a 5′-cap structure and a 3′ poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3′ untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of Nicotiana benthamiana protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect N. benthamiana plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3′ UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.

We selected from landscape phage library probes that bind preferentially Salmonella typhimurium cells compared with other Enterobacteriaceae. The specificity of the phage probes for S. typhimurium was analyzed by the phage-capture test, the enzyme-linked immunosorbent assay (ELISA), and the precipitation test. Interaction of representative probes with S. typhimurium was characterized by fluorescence-activated cell sorting (FACS), and fluorescent, optical and electron microscopy. The results show that the landscape phage library is a rich source of specific and robust probes for S. typhimurium suitable for long-term use in continuous monitoring devices and biosorbents.

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.

The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail. The coat protein accumulation level of the mutant, BaMV40A/DeltaABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana. This suggested that ABC domain might play an important role in BaMV RNA replication. To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40A/DeltaA, -/DeltaB, and -/DeltaC each lacking stem-loop A, B, and C, respectively, were created. Our results showed that accumulation of viral products of mutants BaMV40A/DeltaB and -/DeltaC were not as efficient as wild-type. On the contrary, level of accumulation of viral products of BaMV/DeltaA was similar to that of wild-type in protoplasts and inoculated leaves. Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation. Using UV cross-linking and competition experiments, it was demonstrated that the E. coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain.

This paper presents a method that combines surface swab sampling and wireless biosensing for rapid detection of foodborne pathogens on solid surfaces. The method can be applied to any surfaces, including the surface of food processing tables and fresh produce, for improving food safety. Surface swab sampling is an inexpensive and easy-to-use method of collecting pathogens. A desired area or the whole area of a target surface (if needed) can be swabbed to collect pathogens rapidly. In this study, we first investigated the efficiency of capture and release of a model pathogen, Salmonella Typhimurium, in swab sampling. A Salmonella-contaminated surface (2 × 2 cm2 flat polyethylene slab) was prepared by using a spot-inoculation method. Five drops of a Salmonella solution (20 ul/drop at concentrations from 5X108 cfu/ml to 5X103 cfu/ml) were inoculated on the polyethylene surface and allowed to dry in a biosafety cabinet for 2 hours. Swab sampling was then conducted at a controlled temperature and humidity to capture the pathogen. Finally, the swabs used were dipped in a TBS solution for releasing the captured Salmonella cells. Various swab tip materials were tested, including cotton, rayon, and nylon. The effects of swab force and sonication/vortexing on the capture and release efficiency were also investigated. The released Salmonella cells were finally detected and quantified instantaneously by using a wireless phage-based magnetoelastic biosensor. By comparing the quantities of the initially inoculated cells and detected cells, the efficiency of capture and release was calculated.

This paper investigates a rapid method of detecting bacterial pathogens on 2D food, such as leafy greens. The method is based on our previous work that uses freestanding phage-coated magnetoelastic (ME) biosensors and a surface-scanning coil detector for direct bacterial detection on the surface of food without commonly used sample preparation steps (washing, concentration, enrichment, etc). The ME biosensors used in this work were ferromagnetic alloy strips coated with E2 phage that specifically binds with Salmonella Typhimurium. First, the biosensors (1 mm × 0.2 mm × 30 µm) were placed in rectangular, indexed cavities (1.1 mm × 0.22 mm × 20 µm) that are etched in a glass wafer. A sheet magnet was added to the back of the wafer to hold the sensors in place. The glass wafer with the biosensors and sheet magnet was then flipped and lowered onto Salmonella-tainted spinach leaves. At this time, a weight was added such that the leaves got flattened to improve the chance for the biosensors to physically contact with Salmonella cells. Various weights ranging from 0 g to 120 g were tested to investigate their effects on the Salmonella binding. Measurement sensors loaded with E2 phage and control sensors devoid of the phage were prepared and used. The control sensors served as the reference to compensate for the environmental effects such as temperature and humidity. The biosensors were placed on the surface of the spinach leaves for 2 mins for the binding to occur. The degree of the binding of Salmonella to the measurement sensors was determined by measuring the resonant frequency shift of the sensors using a surface-scanning detection system and confirmed by optical microscopy. The binding of Salmonella was found to increase as the spiked Salmonella concentration increased (103 cfu/ml to 108 cfu/ml). In addition, significant resonant frequency shifts of the measurement sensors (4,800 ± 160 Hz) were observed when 60 g of weight was added. However, further increases in weight did not improve the Salmonella binding with E2 phage. Rather, heavier weights increased the probability of non-specific attachment of Salmonella cells and other residuals from the leaf surface.

Foodborne illness is a common public health problem because food can be contaminated with pathogens at any point in the farm-to-table continuum. This paper presents a rapid method of detecting foodborne bacteria in a small volume of liquid (1 ml) using magnetoelastic (ME) biosensors and a surface-scanning coil detector. The ME biosensors are mass-sensitive, strip-shaped magnetic particles (1 mm × 0.2 mm × 0.03 mm). Phage was coated on the surface of the biosensors to capture a specific pathogen. The resonant frequency of the biosensors decreases when the specific pathogen is attached to the sensor surface. This decrease in resonant frequency was measured in real time by using a surface-scanning coil detector connected to a network analyzer. In this work, the detection device was comprised of the surface-scanning coil detector and glass capillary tubes with two different diameters (0.5 mm and 0.3 mm). The two capillary tubes were nested each other (i.e., the 3 mm tube inserted into the 5 mm tube) to form a flow channel through which a 1 ml bacteria solution was passed. An ME biosensor was placed inside the flow channel through the 0.5 mm tube and positioned where it meets the tip of the 3 mm tube. The 3 mm tube tip served as a stopper to fix the biosensor position in a liquid stream. The surface-scanning detector was positioned under the biosensor for real-time resonant frequency measurement. The flow channel and surface-scanning detector were embedded and fixed in polydimethylsiloxane (PDMS) to ensure the mechanical stability of the entire device. In order to control and change the flow rate of the bacteria solution, a peristatic pump was used to deliver the solution into the flow channel. The E2 phage was used to specifically capture Salmonella Typhimurium cells in a 1 ml concentrated solution passing through the flow channel. The concentration of Salmonella solutions ranged from 103 cfu/ml to 108 cfu/ml. ME biosensors without E2 phage were used as control sensors, and ones coated with E2 phage were used as measurement sensors. Both groups of sensors were tested by passing the Salmonella solutions, and changes in the resonant frequency of the sensors were recorded. The resonant frequency of the measurement sensors were found to decrease, resulting from specific binding of Salmonella on the biosensors. In addition, the resonant frequency shifts of the measurement sensors were much larger than those of the control sensors for the whole tested range of Salmonella concentrations. The presented method can be combined with surface-sampling of bacterial pathogens by swabbing and used for real-time pathogen detection.

Magnetoelastic (ME) biosensors with phage-displayed oligopeptide probes have been demonstrated to be highly successful in rapid detection of various pathogens, including Salmonella enterica, on the fruit and vegetable surfaces. However, in lieu of testing each produce individually, it is advantageous to detect pathogens in the produce wash water for high-throughput analysis. The sanitizing guidelines of the United States Food and Drug Administration (FDA) suggest adding Clorox and chlorine dioxide to the produce wash water in processing plants to disinfect harmful food-borne pathogens on fruit and vegetable surfaces. Therefore, to determine the efficacy of our ME biosensors in testing for food-borne pathogens in produce wash water, we assessed the stability of our biosensors in the presence of Clorox and chlorine dioxide. Specifically, Enzyme Linked Immunosorbent Assay (ELISA) was used to study the potential effect of Clorox and chlorine dioxide on the S. Typhimurium capturing ability of our phage probe. At concentrations recommended by the FDA for Clorox (100 ppm free chlorine content) and by the United States Environmental Protection Agency for chlorine dioxide (4 ppm), we observed no negative effect of these chemicals on the stability and S. Typhimurium capturing ability of our phage-displayed oligopeptide probe. These data demonstrate the potential efficacy of using our ME biosensors with phage-displayed oligopeptide probes in produce wash waters to determine the presence of food-borne pathogens.

This paper presents a filtration method to capture, concentrate, and isolate small quantities of bacterial pathogens in large volumes of liquid streams. The filter used in this work is a multi-layered planar arrangement of phage-coated, strip-shaped magnetic particles (4 mm × 0.8 mm × 0.03 mm) through which a liquid of interest flows. These magnetic particles are held and arranged by a magnetic force produced from solenoid coils. This “phage filter” is designed to capture specific bacterial pathogens through phage-based biomolecular recognition and allow non-specific targets to pass. An advantage of the phage filter is to eliminate a common clogging issue, which often occurs in conventional bead filters. The phage filter consists of a support frame, solenoid coils coupled to the support frame, and a plurality of phage-coated magnetic particles. The support frame is fabricated from a soft magnetic material. The solenoid is configured to generate a defined magnetic field. The phage-coated magnetic particles, magnetically coupled to the support frame, are arranged in a planar array, positioned within the opening of the support frame. Each magnetic particle is fixed at one end and can swing freely at the other end, allowing large, non-specific debris to pass. ANSYS-Maxwell magnetic simulations were run to obtain the magnetic flux vectors and field strength around the filter with varying electric currents and numbers of, coil layers and turns. The results were compared to experimental results produced using iron powders and a gaussmeter, followed by the selection of the optimal filter design. Then, a series of experiments were conducted where liquid solutions containing different concentrations of Salmonella were passed through the filter, and the capture efficiency was calculated by plate counting. The biomolecular recognition filter can be combined with standard detection methods, such as qPCR, to detect and identify pathogens rapidly.

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.

Mandarin ‘one’-phrase, [yi ‘one’+classifier+noun], has multiple interpretations, such as counting /measuring phrases, negative polarity items (NPIs), and expressions meaning ‘whole’. Each function of the ‘one’-phrase is treated as a construction that has different relationships among its three components. ‘One’ serves as a reference point on a scale, so the numeral sequence can mean maximality or minimality depending on contexts. The scalar implications are built into the whole construction instead of one lexeme through grammaticalization. This study aims to provide a unified account for the synchronic polysemous ‘one’-phrase by looking into its diachronic development.

Proceedings of the 37th Annual Meeting of the Berkeley Linguistics Society

Brain damage within the right middle cerebral artery (MCA) territory is particularly disruptive to mediolateral postural stabilization. The objective of this cross-sectional study was to test the hypothesis that chronic right MCA infarcts (as compared to left) are associated with slower and more bilaterally asymmetrical gait. We further hypothesized that in those with chronic right MCA infarct, locomotor performance is more dependent on gray matter (GM) volumes within noninfarcted regions of the brain that are involved in motor control yet lie outside of the MCA territory.Gait speed was assessed in 19 subjects with right MCA infarct, 20 with left MCA infarct, and 108 controls. Bilateral plantar pressure and temporal symmetry ratios were calculated in a subset of the cohort. GM volumes within 5 regions outside of the MCA territory (superior parietal lobe, precuneus, caudate, putamen, and cerebellum) were quantified from anatomic MRIs.Right and left infarct groups had similar poststroke duration (7.6 ± 6.0 years), infarct size, and functional independence. The right infarct group demonstrated slower gait speed and greater asymmetry compared to the left infarct group and controls (p0.05). In the right infarct group only, those with larger GM volumes within the cerebellum (r(2) = 0.32, p = 0.02) and caudate (r(2) = 0.56, p0.001) exhibited faster gait speed.Individuals with chronic lesions within the right MCA territory, as compared to the left MCA territory, exhibit slower, more asymmetrical gait. For these individuals, larger GM volumes within regions outside of the infarcted vascular territory may help preserve locomotor control.

This study aims to investigate abstract reasoning and embodied cognition through the analysis of image schemas and conceptual metaphors in the interplay of art and language. Chinese calligraphy is noteworthy due to its unique embodied characteristics and image-schematic representations of visual art and language. The art of Chinese calligraphy not only represents the visual forms of Chinese characters but also conveys meanings, emotion, and style, demonstrating the aesthetics of language and art. By analyzing image schemas and metaphors in classical works of art, this paper shows how semantics is conceptualized and embodied through visual representation of Chinese calligraphy. In this study, we examine how semantics is visualized within the topological structure of cognitive mechanisms of a CONTAINER schema, the crucial image schema that structures the conceptualization of spatial relation concepts. This paper proposes that the CONTAINER schema, the BALANCE schema, the FORCE schema, as well as the metaphors SIGNIFICANCE IS SIZE and MIND IS A BODY, which may motivate the calligrapher’s creative process, underlie the art of Chinese calligraphy.

Objective. To present our experience with single mini-incision complete urinary tract exenteration (CUTE) for female dialysis patients suffering from urothelial carcinoma (UC).Patients and Methods. Institutional review board approval was obtained. From 2005 through 2012, 14 female dialysis patients with UC underwent single mini-incision CUTE, in combination with radical hysterectomy and bilateral salpingo-oophorectomy. All were placed in the modified dorsal lithotomy position without repositioning. An infraumbilical midline mini-incision was made. Bilateral nephroureterectomy was first performed entirely extraperitoneally, followed by radical cystectomy with removal of the uterus and ovaries transperitoneally.Results. All procedures were done successfully without major complications. The median operative time was 242.5 minutes, and estimated blood loss was 500 mL. The median time to oral intake was 2 postoperative days; the median hospital stay was 11 days. Ten patients remained cancer-free at a median follow-up of 46.5 months; six patients were confirmed as having preoperatively undetectable UC or renal cell carcinoma, even after reviewing preoperative computed tomography.Conclusions. This modified technique provides a time-saving complete urinary tract extirpation to eliminate preoperatively undetectable malignancy, reduce metachronous recurrences, and avert perioperative complications associated with pneumoperitoneum and repositioning. Good cancer control and early convalescence can mutually be achieved in experienced hands.

Background Bamboo mosaic virus (BaMV) and the Potato virus X (PVX) are members of the genus Potexvirus and have a single-stranded positive-sense RNA genome. The 3′-untranslated region (UTR) of the BaMV RNA genome was mapped structurally into ABC (a cloverleaf-like), D (a stem-loop), and E (pseudoknot) domains. The BaMV replicase complex that was isolated from the infected plants was able to recognize the 3′ UTR of PVX RNA to initiate minus-strand RNA synthesis in vitro. Results To investigate whether the 3′ UTR of PVX RNA is also compatible with BaMV replicase in vivo, we constructed chimera mutants using a BaMV backbone containing the PVX 3′ UTR, which was inserted in or used to replace the various domains in the 3′ UTR of BaMV. None of the mutants, except for the mutant with the PVX 3′ UTR inserted upstream of the BaMV 3′ UTR, exhibited a detectable accumulation of viral RNA in Nicotiana benthamiana plants. The in vitro BaMV RdRp replication assay demonstrated that the RNA products were generated by the short RNA transcripts, which were derived from the chimera mutants to various extents. Furthermore, the Vmax/KM of the BaMV 3′ UTR (rABCDE) was approximately three fold higher than rABCP, rP, and rDE in minus-strand RNA synthesis. These mutants failed to accumulate viral products in protoplasts and plants, but were adequately replicated in vitro. Conclusions Among the various studied BaMV/PVX chimera mutants, the BaMV-S/PABCDE that contained non-interrupted BaMV 3′ UTR was the only mutant that exhibited a wild-type level of viral product accumulation in protoplasts and plants. These results indicate that the continuity of the domains in the 3′ UTR of BaMV RNA was not interrupted and the domains were not replaced with the 3′ UTR of PVX RNA in vivo.

Pemphigus vulgaris (PV) is an autoimmune disease in which the autoantibody, immunoglobulin G, is directed against the keratinocytes in the epidermis. The classic presentations of PV are flaccid vesicles or bullae over the oral mucosa, trunk, groin, and extremities. The age of onset is usually between 40 and 60 years, and cases of PV in children or adolescent patients are rare. Here, we present a 17-year-old boy who had painful oral ulcers for 3 months initially and bullae spreading to the whole body in the following days. Paraneoplastic pemphigus was another differential diagnosis due to the atypical appearance of the skin lesion. However, PV was confirmed by hematoxylin and eosin staining and immunofluorescence examination of the skin biopsy specimens. The patient had a good response to corticosteroid treatment and the immunosuppressive agent, rituximab.

Current techniques used to concentrate pathogens, such as centrifugation and size-exclusion filtration, are non-specific for pathogens of interest and inefficient to deal with large volumes of liquids and liquid foods. In addition, the isolated pathogens need to be analyzed subsequently with expensive, laborious, sample-based lab methods (e.g., culturing and PCR) that do not enable rapid identification of the pathogens. This paper presents a revolutionary method for the capture and rapid detection of small amounts of specific bacteria from entire liquid streams. The method combines a volumetric biomolecular filter and an automated biosensor measurement system. The volumetric biomolecular filter is a three-dimensional, multi-layered net with each layer made up of hundreds of phage-coated magnetoelastic (ME) biosensors with a pre-determined resonant frequency. These biosensors are arrayed with a desired spacing and held flexibly (i.e., cantilever-like configuration) on an electro-magnetized silicon steel comb structure. The phage that is coated on the biosensors are responsible for capturing a specific pathogen. In this way, only the pathogen of interest can be captured on the filter whereas the other matters (non-specific pathogens, debris, etc.) are allowed to pass. In addition, the biosensors can bend over for large, heavy debris, eliminating a common issue of clogging found in the current filtration techniques. Once the entire liquid is flowed over, the biosensors on the filter are transferred into etched cavities on a silicon wafer by shutting off the electromagnetic field. These etched cavities are indexed such that a surface-scanning detector can be used to wirelessly measure the biosensors sequentially in an automated manner. The resonant frequency changes of the biosensors due to the attachment of the pathogen can be measured in 250 milliseconds per sensor. The presented method enables the evaluation of large volumes of liquids for the presence of small numbers of pathogens. The capture efficiency of pathogens was found to be dependent on the filter design.

This paper presents a rapid method of detecting live versus dead pathogenic bacteria. The method combines phage-coated magnetoelastic (ME) biosensors and a wireless planar coil detector, enabling real-time monitoring of the growth and viability of specific bacteria in a nutrient broth. The ME biosensor used in this investigation is composed of a strip-shaped ME resonator (1 mm x 0.2 mm x 30 um) upon which a landscape phage is coated to capture specific bacteria. E2 phage with high binding affinity for Salmonella Typhimurium was used as a model study. The specificity of E2 phage has been reported to be 200 in 108 background bacteria. When the binding of Salmonella occurs, the mass of the biosensor increases, which simultaneously results in a decrease in the biosensor's resonant frequency. Monitoring of this mass-induced resonant frequency change allows for the instantaneous detection and quantification of Salmonella. ME biosensors with a pre-determined resonant frequency were first exposed to a low-concentration Salmonella suspension to capture a small but detectable amount of the bacteria on the sensor surface. The wireless coil detector was then used to measure the resonant frequency changes of the biosensors as a function of time in a nutrient broth. Live cells can grow, while dead ones can't, which leads to distinct differences in the resonant frequency changes over time. Hence, this methodology offers direct, real-time detection, quantification, and viability determination of specific bacteria.

Previous evidence has indicated that the P25 protein encoded by Potato virus X (PVX) inhibits either the assembly or function of the effector complexes in the RNA silencing-based antiviral defence system (Bayne et al., Cell-to-cell movement of Potato Potexvirus X is dependent on suppression of RNA silencing. Plant J.44, 471-482). This finding prompted us to investigate the possibility that P25 targets the Argonaute (AGO) effector nuclease of RNA silencing. Co-immunoprecipitation and Western blot analysis indicated that there is a strong interaction between P25 and AGO1 of Arabidopsis when these proteins are transiently co-expressed in Nicotiana benthamiana. P25 also interacts with AGO1, AGO2, AGO3 and AGO4, but not with AGO5 and AGO9. As an effective suppressor, the amount of AGO1 accumulated in the presence of P25 was dramatically lower than that infiltrated with HcPro, but was restored when treated with a proteasome inhibitor MG132. These findings are consistent with the idea that RNA silencing is an antiviral defence mechanism and that the counter-defence role of P25 is through the degradation of AGO proteins via the proteasome pathway. Further support for this idea is provided by the observation that plants treated with MG132 are less susceptible to PVX and its relative Bamboo mosaic virus.

Food borne illnesses from the ingestion of S. typhimurium have been of primary concern due to their common occurrence in food products of daily consumption. In this paper, micron size, magnetoelastic (ME) biosensors for the detection of S. typhimurium were fabricated and tested in liquid solutions containing known concentrations of S. typhimurium cells. The biosensors are comprised of a ME sensor platform and immobilized bio-molecular recognition element (E2 phage) that has been engineered to bind the S. typhimurium multi-valently. The micron size ME sensor platforms were manufactured using microelectronics fabrication techniques. Phage was engineered at Auburn University and immobilized onto all surfaces of the sensor. The ME biosensor oscillates with a characteristic resonance frequency when subjected to a time varying magnetic field. Binding between the phage and bacteria, adds mass to the sensor that causes a shift in the sensor's resonance frequency. Sensors with the dimension of 500x100x4 μm were exposed to S. typhimurium with increasing known concentrations ranging from 5 x10 1 to 5 x 10 7 cfu/ml. The ME biosensor exhibited high sensitivity and a detection limit better than 50 cfu/ml.

Lin K-C, Chung H-Y, Wu C-Y, Liu H-L, Hsieh Y-W, Chen I-H, Chen C-L, Chuang L-L, Liu J-S, Wai Y-Y: Constraint-induced therapy versus control intervention in patients with stroke: a functional magnetic resonance imaging study. Am J Phys Med Rehabil 2010;89:177‐185. Objective: This study compared the effects of a distributed form of constraint-induced therapy with control intervention in motor recovery and brain reorganization after stroke. Design: A two-group randomized controlled trial with pretreatment and posttreatment measures was conducted. Thirteen patients with stroke were randomly assigned to the distributed form of constraint-induced therapy (n 5) or the control intervention group (n 8). Outcome measures included the Fugl-Meyer Assessment, the Motor Activity Log, and functional magnetic resonance imaging examination. The number of activation voxels and laterality index were determined from the functional magnetic resonance imaging data for the study of brain reorganization. Results: The distributed form of constraint-induced therapy group exhibited significantly greater improvements in the Fugl-Meyer Assessment and Motor Activity Log than the control intervention group. The functional magnetic resonance imaging data showed that distributed form of constraintinduced therapy significantly increased activation in the contralesional hemisphere during movement of the affected and unaffected hand. The control intervention group showed a decrease in primary sensorimotor cortex activation of the ipsilesional hemisphere during movement of the affected hand. Conclusions: The preliminary findings indicate that brain adaptation may be modulated by specific rehabilitation practices, although generalization of the functional magnetic resonance imaging findings is limited by sample size. Further research is needed to identify the specific neural correlates of the behavioral gains achieved after rehabilitation therapies.

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.

Although immunodeficiency is usually considered a prerequisite of oncogenesis, a detailed immune pro- file in cancer has not yet been described. Without such profiling, it is not surprising that there is a vast discrepancy in the responses of cancer patients to immunotherapy. Our results show that the integrity of the immune system deteriorates with cancer progression by displaying a trend toward decreasing levels of functional T cells, including CD4, naive, and central memory T cells, and an expansion of hyporesponsive populations such as CD28⁻ and CMV-specific T cells. One hundred and one patients constitute the study group for the observational study reported in this paper. Forty-eight patients with newly diagnosed stages III and IV and 53 patients with extensively treated stage IV disease. The costimulatory molecules CD27 and CD28 were downregulated in all patients. Among the proinflammatory cytokines (IL-6, TNF-α, IFN-γ), only IL-6 differed significantly among the groups, increasing as the cancer stage progressed. Plasma IL-7 did not diVer among the participants. The relative deficits of naive T cells in cancer patients may be associated with the downregulation of IL-7Rα expression rather than changes in the circulating levels of IL-7. The downregulation of IL-7Rα expression was shown to be associated with increased levels of intracellular CMV. The present study suggests that the immune impairment in patients with cancer is associated with multiple factors, such as the stage of cancer, consequence of CMV infection and impact of treatment.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that patrol tissues to sense danger signals and activate specific immune responses. In addition, they also play a role in inflammation and tissue repair. Here, we show that oxygen availability is necessary to promote full monocyte-derived DC differentiation and maturation. Low oxygen tension (hypoxia) inhibits expression of several differentiation and maturation markers (CD1a, CD40, CD80, CD83, CD86, and MHC class II molecules) in response to lipopolysaccharide (LPS), as well as their stimulatory capacity for T-cell functions. These events are paralleled by impaired up-regulation of the chemokine receptor CCR7, an otherwise necessary event for the homing of mature DCs to lymph nodes. In contrast, hypoxia strongly up-regulates production of proinflammatory cytokines, particularly TNFα and IL-1β, as well as the inflammatory chemokine receptor CCR5. Subcutaneous injection of hypoxic DCs into the footpads of mice results in defective DC homing to draining lymph nodes, but enhanced leukocyte recruitment at the site of injection. Thus, hypoxia uncouples the promotion of inflammatory and tissue repair from sentinel functions in DCs, which we suggest is a safeguard mechanism against immune reactivity to damaged tissues.

This paper presents a multiple magnetoelastic (ME) biosensor system for in-situ detection of S. typhimurium and B. anthracis spores in a flowing bacterial/spore suspension (5 x 101 - 5 x 108 cfu/ml). The ME biosensor was formed by immobilizing filamentous phage (specific to each detection target) on the ME platforms. An alternating magnetic field was used to resonate the ME biosensor to determine its resonance frequency. When cells/spores are bound to a ME biosensor surface, the additional mass of the cells/spores causes a decrease in the resonance frequency of the biosensor. The detection system was composed of a control sensor, an E2 phage-based biosensor (specific to S. typhimurium) and a JRB7 phage-based biosensor (specific to B. anthracis spores). 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 observed to be 5 x 103 cfu/ml. The results show that phage-based ME biosensors can detect multiple pathogens simultaneously and offer good performance, including good sensitivity 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.

A new diagnostic probe selected from a landscape phage library was used in combination with a free standing magnetostrictive platform to form a wireless biosensor with quick response and high accuracy. The immobilization of the phage-derived probes leads to a 3D biomolecular recognition layer that captures the target spores multivalently. After the phage-coated biosensors were exposed to suspensions of the target spores, the binding of spores to the sensors resulted in a decrease of the resonant frequency due to the additional mass of the attached spores. Scanning electron microscopy was used to relate the observed frequency changes to the actual number of spores bound to the sensor

Background: Recent use of Bacillus anthracis spores as a bioweapon has highlighted the need for a continuous monitoring system. Current monitoring systems rely on antibody-derived probes, which are not hardy enough to withstand long-term use under extreme conditions. We describe new, phage-derived probes that can be used as robust substitutes for antibodies.Methods: From a landscape phage library with random octapeptides displayed on all copies of the major phage coat protein of the phage fd-tet, we selected clones that bound to spores of B. anthracis (Sterne strain). ELISA, micropanning, and coprecipitation assays were used to evaluate the specificity and selectivity with which these phage bound to B. anthracis spores.Results: Peptides on the selected clones directed binding of the phage to B. anthracis spores. Most clones exhibited little or no binding to spores of distantly related Bacillus species, but some binding was observed with spores of closely related species. Our most specific spore-binding phage displayed a peptide EPRLSPHS (several thousand peptides per phage) and bound 3.5- to 70-fold better to spores of B. anthracis Sterne than to spores of other Bacillus species.Conclusions: The selected phage probes bound preferentially to B. anthracis Sterne spores compared with other Bacillus species. These phage could possibly be further developed into highly specific and robust probes suitable for long-term use in continuous monitoring devices and biosorbents.

Classical swine fever virus (CSFV) causes a broad range of disease in pigs, from acute symptoms including high fever and hemorrhages, to chronic disease or unapparent infection, depending on the virus strain. CSFV belongs to the genus Pestivirus of the family Flaviviridae. It carries a single-stranded positive-sense RNA genome. An internal ribosomal entry site (IRES) in the 5' untranslated region (UTR) drives the translation of a single open reading frame encoding a 3898 amino acid long polypeptide chain. The open reading frame is followed by a 3' UTR comprising four highly structured stem-loops. In the present study, a synthetic RNA composed of the 5' and 3' UTRs of the CSFV genome devoid of any viral coding sequence and separated by a luciferase gene cassette (designated 5'UTR-Luc-3'UTR) triggered apoptotic cell death as early as 4 h post-transfection. The apoptosis was measured by DNA laddering analysis, TUNEL assay, annexin-V binding determined by flow cytometry, and by analysis of caspase activation. Contrasting with this, only trace DNA laddering was observed in cells transfected with the individual 5' or 3' UTR RNA; even when the 5' UTR and 3' UTR were co-transfected as separate RNA molecules, DNA laddering did not reach the level induced by the chimeric 5'UTR-Luc-3'UTR RNA. Interestingly, RNA composed of the 5'UTR and of stem-loop I of the 3'UTR triggered much stronger apoptosis than the 5' or 3'UTR alone. These results indicate that the 5' and 3' UTRs act together in cis induce apoptosis. We furthered obtained evidence that the UTR-mediated apoptosis required double-stranded RNA and involved translation shutoff possibly through activation of PKR.

Objective: To determine whether DAZL1 is expressed in human fetal ovarian tissue. Design: The presence of DAZL1 expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Setting: Academic tertiary care medical center and research unit of university. Patient(s): Five female abortuses between the 19th and 22nd week of gestational age. Intervention(s): Fetal ovarian tissues were collected immediately after the cessation of the heart beat. Main Outcome Measure(s): The product of RT-PCR. Result(s): DAZL1 expression was detected in all five samples. Conclusion(s): DAZL1 is not only expressed in human testes but also in ovaries. It may play a role in germ cell survival and gonad development in both sexes.

To investigate the plant genes affected by Bamboo mosaic virus (BaMV) infection, we applied a cDNA-amplified fragment length polymorphism technique to screen genes with differential expression. A serine/threonine kinase-like (NbSTKL) gene of Nicotiana benthamiana is upregulated after BaMV infection. NbSTKL contains the homologous domain of Ser/Thr kinase. Knocking down the expression of NbSTKL by virus-induced gene silencing reduced the accumulation of BaMV in the inoculated leaves but not in the protoplasts. The spread of GFP-expressing BaMV in the inoculated leaves is also impeded by a reduced expression of NbSTKL. These data imply that NbSTKL facilitates the cell-to-cell movement of BaMV. The subcellular localization of NbSTKL is mainly on the cell membrane, which has been confirmed by mutagenesis and fractionation experiments. Combined with the results showing that active site mutation of NbSTKL does not change its subcellular localization but significantly affects BaMV accumulation, we conclude that NbSTKL may regulate BaMV movement on the cell membrane by its kinase-like activity. Moreover, the transient expression of NbSTKL does not significantly affect the accumulation of Cucumber mosaic virus (CMV) and Potato virus X (PVX); thus, NbSTKL might be a specific protein facilitating BaMV movement.

not Available.

We thank Dr. Derhovanessian for the comments on our article ‘‘Immune impairment in patients with terminal cancers: influence of cancer treatments and cytomegalovirus infection’’ pointing out an important yet less studied role of CMV in modulating immune status in patients with cancer. Theoretically, we also believe that CMV serostatus could be an useful marker in predicting a patient’s immune outcome following treatment and disease progression. In the early phase of enrollment, we had 65 participants being tested for their CMV serostatus (14 normal; 13 stage I treatment-naive; 20 stage III/IV treatment-naive; 18 stage IV heavily treated). Only 2 patients in stage I were CMV IgG-negative and the other 63 were positive. It was therefore practically impossible to enroll a sufficient number of sero-negative patients to demonstrate the power of CMV serostatus. Our data suggested that a number of factors including stage of disease, treatment and CMV reactivation could contribute to the deviation of immune status in stage IV heavily treated patients from stage III/IV treatment-naive patients. While we strongly believe that repetitive treatment-associated CMV reactivation is the main factor exhausting their immune integrity in cancer patients, we were unable to demonstrate this based on current data. Indeed, the amount of CMV-specific T cells was not different between stage I treatment-naive patients (not included in the current article) and stage III/IV treatmentnaive patients, supporting the notion that CMV-associated immune signatures may well be a consequence of treatments. On the other hand, if when we compared those stage I treatment-naive patients with a normal group and those stage III/IV treatment-naive patients, some stage-associated differences in immune status were found; for instance CD27, CD127 in naive populations and IL-6. Therefore, the impact of CMV infection on immune status may not be the sole factor compromising immune functions in patients with cancer. Still, among these factors, we may be in a better position to control CMV than cancer. One way to identify the impact of CMV infection on immune status is to study patients with pre-emptive therapy during chemotherapy. Our unpublished data show that some of the immune parameters can be reversed in vitro in PBMC from stage IV patients by treatment with ganciclovir. The impact of CMV infection in patients with cancer during treatment somehow looks like accelerating the process of immune senescence. Whether pre-emptive treatment will be helpful in preserving patients’ immunity is not known at this time, but our data encourage the development of strategies to control CMV reactivation.