Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

BACKGROUND: Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

Background: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phageresistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD 50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophag

Optimization of Urease Production Capacity of a Novel Salt-Tolerant <i>Staphylococcus</i><i>xylosus</i> Strain through Response Surface Modeling

Encouraging advances have been made in the application of microbial mineralization towards fixing and improving desertified sandy soils. However, desert soils in arid areas exhibit high salinity that may limit urease activity and production in microbial strains, thereby affecting the solidification effects of microbial calcium binders in saline soils. In this study, a salt-tolerant microbial strain (A80) that produced urease was identified from saline soils of the Qaidam Basin. The culture conditions of the strain were optimized using single-factor tests and response surface methods to optimize urease yields and activity. The optimal composition of the A80 medium included an inoculation amount of 6.32% (V/V), a yeast extract powder concentration of 15.43 g/L, a glucose concentration of 5.20 g/L, a salinity of 3%, and an incubation temperature of 36 °C. Urease activity increased by 64.80% after using optimized medium. The A80 microbial calcium-cementing agent was also used to solidify saline soils, leading to an increased unconfined compressive strength of the solidified saline soil by 25.70%. Thus, the optimization method resulted in improvements in the cultivation of a salt-tolerant strain

Optimization of Urease Production Capacity of a Novel Salt-Tolerant Staphylococcusxylosus Strain through Response Surface Modeling

Encouraging advances have been made in the application of microbial mineralization towards fixing and improving desertified sandy soils. However, desert soils in arid areas exhibit high salinity that may limit urease activity and production in microbial strains, thereby affecting the solidification effects of microbial calcium binders in saline soils. In this study, a salt-tolerant microbial strain (A80) that produced urease was identified from saline soils of the Qaidam Basin. The culture conditions of the strain were optimized using single-factor tests and response surface methods to optimize urease yields and activity. The optimal composition of the A80 medium included an inoculation amount of 6.32% (V/V), a yeast extract powder concentration of 15.43 g/L, a glucose concentration of 5.20 g/L, a salinity of 3%, and an incubation temperature of 36 &deg;C. Urease activity increased by 64.80% after using optimized medium. The A80 microbial calcium-cementing agent was also used to solidify saline soils, leading to an increased unconfined compressive strength of the solidified saline soil by 25.70%. Thus, the optimization method resulted in improvements in the cultivation of a salt-tolerant strain

The Horizontal Bearing Capacity of Composite Concrete-Filled Steel Tube Piles

Steel casings (SCs) are extensively and increasingly used to stabilize the borehole wall in the construction of bridge pile foundations. Steel casings (SCs), together with reinforced concrete piles (RCPs), form composite concrete-filled steel tube piles (CCFSTPs), which differ significantly from ordinary RCPs in horizontal bearing capacity. In this study, based on the characteristics of CCFSTPs, the horizontal bearing capacity of a CCFSTP was examined through a centrifugal model test with the length of the steel casing (LSC) and the modulus of the soil mass in the steel casing soil compaction zone (ESCSC_zone) as variables. Pile-side soil resistance, load-displacement curves, and pile moment curves were obtained for the CCFSTP. The results show that increasing LSC within a range of 12 cm significantly increases the ultimate horizontal bearing capacity of the CCFSTP, and further increasing LSC beyond 12 cm produces a continuous increase in the ultimate horizontal bearing capacity of the CCFSTP but only to an insignificant extent. In addition, increasing ESCSC_zone increases the ultimate horizontal bearing capacity of the CCFSTP, but to a relatively small extent. The results of this study provide a theoretical basis and technical support for the design and construction of CCFSTPs

Bisphenol A Exacerbates Allergic Inflammation in an Ovalbumin-Induced Mouse Model of Allergic Rhinitis

Purpose. Bisphenol A (BPA) is found in many plastic products and is thus a common environmental endocrine disruptor. Plastic-related health problems, including allergic diseases, are attracting increasing attention. However, few experimental studies have explored the effect of BPA on allergic rhinitis (AR). We explore whether BPA was directly related to the allergic inflammation induced by ovalbumin (OVA) in AR mice. Methods. We first constructed OVA-induced mouse model, and after BPA administration, we evaluated nasal symptoms and measured the serum OVA-specific IgE levels by ELISA. Th2 and Treg-related cytokines of nasal mucosa were measured by cytometric bead array. Th2 and Treg-specific transcription factor levels were assayed by PCR. The proportions of CD3+CD4+IL-4+Th2 and CD4+Helios+Foxp3+ T cells (Tregs) in spleen tissue were determined by flow cytometry. Results. Compared to OVA-only-induced mice, BPA addition increased nasal symptoms and serum OVA-specific IgE levels. OVA and BPA coexposure significantly increased IL-4 and IL-13 protein levels compared to those after OVA exposure alone. BPA plus OVA tended to decrease the IL-10 protein levels compared to those after OVA alone. Coexposure to OVA and BPA significantly increased the GATA-3-encoding mRNA level, and decreased the levels of mRNAs encoding Foxp3 and Helios, compared to those after OVA exposure alone. BPA increased the Th2 cell proportion, and decreased that of Tregs, compared to the levels with OVA alone. Conclusion. BPA exerted negative effects by exacerbating AR allergic symptoms, increasing serum OVA-specific IgE levels, and compromising Th2 and Treg responses

Effects of <em>Staphylococcus aureus</em> Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In Vitro

A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it

<i>OsSPLs</i> Regulate Male Fertility in Response to Different Temperatures by Flavonoid Biosynthesis and Tapetum PCD in PTGMS Rice

Photoperiod and thermo-sensitive genic male sterile (PTGMS) rice is an important resource for two line hybrid rice production. The SQUAMOSA–promoter binding, such as the (SPL) gene family, encode the plant specific transcription factors that regulate development and defense responses in plants. However, the reports about SPLs participating in male fertility regulation are limited. Here, we identified 19 OsSPL family members and investigated their involvement in the fertility regulation of the PTGMS rice lines, PA2364S and PA2864S, with different fertility transition temperatures. The results demonstrated that OsSPL2, OsSPL4, OsSPL16 and OsSPL17 affect male fertility in response to temperature changes through the MiR156-SPL module. WGCNA (weighted gene co-expression network analysis) revealed that CHI and APX1 were co-expressed with OsSPL17. Targeted metabolite and flavonoid biosynthetic gene expression analysis revealed that OsSPL17 regulates the expression of flavonoid biosynthesis genes CHI, and the up regulation of flavanones (eriodictvol and naringenin) and flavones (apigenin and luteolin) content contributed to plant fertility. Meanwhile, OsSPL17 negatively regulates APX1 to affect APX (ascorbate peroxidase) activity, thereby regulating ROS (reactive oxygen species) content in the tapetum, controlling the PCD (programmed cell death) process and regulating male fertility in rice. Overall, this report highlights the potential role of OsSPL for the regulation of male fertility in rice and provides a new insight for the further understanding of fertility molecular mechanisms in PTGMS rice

A Camera Intrinsic Matrix-Free Calibration Method for Laser Triangulation Sensor

Laser triangulation sensors (LTS) are widely used to acquire depth information in industrial applications. However, the parameters of the components, e.g., the camera, of the off-the-shelf LTS are typically unknown. This makes it difficult to recalibrate the degenerated LTS devices during regular maintenance operations. In this paper, a novel one-dimensional target-based camera intrinsic matrix-free LTS calibration method is proposed. In contrast to conventional methods that calibrate the LTS based on the precise camera intrinsic matrix, we formulate the LTS calibration as an optimization problem taking all parameters of the LTS into account, simultaneously. In this way, many pairs of the camera intrinsic matrix and the equation of the laser plane can be solved and different pairs of parameters are equivalent for displacement measurement. A closed-form solution of the position of the one-dimensional target is proposed to make the parameters of the LTS optimizable. The results of simulations and experiments show that the proposed method can calibrate the LTS without knowing the camera intrinsic matrix. In addition, the proposed approach significantly improves the displacement measurement precision of the LTS after calibration. In conclusion, the proposed method proved that the precise camera intrinsic matrix is not the necessary condition for LTS displacement measurement.Applied Science, Faculty ofNon UBCEngineering, School of (Okanagan)ReviewedFacult