Generating Biologically Stable TNA Aptamers that Function with High Affinity and Thermal Stability.

Aptamers are often prone to nuclease digestion, which limits their utility in many biomedical applications. Here we describe a xeno-nucleic acid system based on α-l-threofuranosyl nucleic acid (TNA) that is completely refractory to nuclease digestion. The use of an engineered TNA polymerase permitted the isolation of functional TNA aptamers that bind to HIV reverse transcriptase (HIV RT) with KD's of ∼0.4-4.0 nM. The aptamers were identified using a display strategy that provides a powerful genotype-phenotype linkage. The TNA aptamers remain active in the presence of nuclease and exhibit markedly higher thermal stability than monoclonal antibodies. The combined properties of biological stability, high binding affinity, and thermal stability make TNA aptamers a powerful system for the development of diagnostic and therapeutic agents.

Thermostable single domain antibody-maltose binding protein fusion for Bacillus anthracis spore protein BclA detection.

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb-A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K(D) ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP-sdAb gave a yield of approximately 100mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb-A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1mg/ml for 1h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1h at 90 °C. The PfuMBP-sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb-A5 both as a capture reagent and as a detection reagent.

Calibrating canines-a universal detector calibrant for detection dogs.

Since the advent of the Universal Detector Calibrant (UDC) by scientists at Florida International University in 2013, this tool has gone largely unrecognized and under-utilized by canine scent detection practitioners. The UDC is a chemical that enables reliability testing of biological and instrumental detectors. Training a biological detector, such as a scent detection canine, to respond to a safe, non-target, and uncommon compound has significant advantages. For example, if used prior to a search, the UDC provides the handler with the ability to confirm the detection dog is ready to work without placing target odor on site (i.e., a positive control), thereby increasing handler confidence in their canine and providing documentation of credibility that can withstand legal scrutiny. This review describes the UDC, summarizes its role in canine detection science, and addresses applications for UDC within scent detection canine development, training, and testing.

A canine model to evaluate the effect of exercise intensity and duration on olfactory detection limits: the running nose.

Detection canines serve critical roles to support the military, homeland security and border protection. Some explosive detection tasks are physically demanding for dogs, and prior research suggests this can lead to a reduction in olfactory detection sensitivity. To further evaluate the effect of exercise intensity on olfactory sensitivity, we developed a novel olfactory paradigm that allowed us to measure olfactory detection thresholds while dogs exercised on a treadmill at two different exercise intensities. Dogs (n = 3) showed a decrement in olfactory detection for 1-bromooctane at 10-3 (v/v) dilutions and lower under greater exercise intensity. Dogs' hit rate for the lowest concentration dropped from 0.87 ± 0.04 when walking at low intensity to below 0.45 ± 0.06 when trotting at moderate intensity. This decline had an interaction with the duration of the session in moderate intensity exercise, whereby dogs performed near 100% detection in the first 10 min of the 8 km/h session, but showed 0% detection after 20 min. Hit rates for high odor concentrations (10-2) were relatively stable at both low (1 ± 0.00) and moderate (0.91 ± 0.04) exercise intensities. The paradigm and apparatus developed here may be useful to help further understand causes of operationally relevant olfactory detection threshold decline in dogs.

Evaluation of canine training aids containment for homemade explosive and components by headspace analysis and canine testing.

While canines are most commonly trained to detect traditional explosives, such as nitroaromatics and smokeless powders, homemade explosives (HMEs), such as fuel-oxidizer mixtures, are arguably a greater threat. As such, it is imperative that canines are sufficiently trained in the detection of such HMEs. The training aid delivery device (TADD) is a primary containment device that has been used to house HMEs and HME components for canine detection training purposes. This research assesses the odor release from HME components, ammonium nitrate (AN), urea nitrate (UN), and potassium chlorate (PC), housed in TADDs. Canine odor recognition tests (ORTs) were used with analytical data to determine the detectability of TADDs containing AN, UN, or PC. Headspace analysis by gas chromatography/mass spectrometry (GC/MS) with solid-phase microextraction (SPME) or online cryotrapping were used to measure ammonia or chlorine, as well as other unwanted odorants, emanating from bulk AN, UN, and PC in TADDs over 28 weeks. The analytical data showed variation in the amount of ammonia and chlorine over time, with ammonia from AN and UN decreasing slowly over time and the abundance of chlorine from PC TADDs dependent on the frequency of exposure to ambient air. Even with these variations in odor abundance, canines previously trained to detect bulk explosive HME components were able to detect all three targets in glass and plastic TADDs for at least 18 months after loading. Detection proficiency ranged from 64% to 100% and was not found to be dependent on either age of material.

Genetic barcodes for improved environmental tracking of an anthrax simulant.

The development of realistic risk models that predict the dissemination, dispersion and persistence of potential biothreat agents have utilized nonpathogenic surrogate organisms such as Bacillus atrophaeus subsp. globigii or commercial products such as Bacillus thuringiensis subsp. kurstaki. Comparison of results from outdoor tests under different conditions requires the use of genetically identical strains; however, the requirement for isogenic strains limits the ability to compare other desirable properties, such as the behavior in the environment of the same strain prepared using different methods. Finally, current methods do not allow long-term studies of persistence or reaerosolization in test sites where simulants are heavily used or in areas where B. thuringiensis subsp. kurstaki is applied as a biopesticide. To create a set of genetically heterogeneous yet phenotypically indistinguishable strains so that variables intrinsic to simulations (e.g., sample preparation) can be varied and the strains can be tested under otherwise identical conditions, we have developed a strategy of introducing small genetic signatures ("barcodes") into neutral regions of the genome. The barcodes are stable over 300 generations and do not impact in vitro growth or sporulation. Each barcode contains common and specific tags that allow differentiation of marked strains from wild-type strains and from each other. Each tag is paired with specific real-time PCR assays that facilitate discrimination of barcoded strains from wild-type strains and from each other. These uniquely barcoded strains will be valuable tools for research into the environmental fate of released organisms by providing specific artificial detection signatures.

Detection and tracking of a novel genetically tagged biological simulant in the environment.

A variant of Bacillus thuringiensis subsp. kurstaki containing a single, stable copy of a uniquely amplifiable DNA oligomer integrated into the genome for tracking the fate of biological agents in the environment was developed. The use of genetically tagged spores overcomes the ambiguity of discerning the test material from pre-existing environmental microflora or from previously released background material. In this study, we demonstrate the utility of the genetically "barcoded" simulant in a controlled indoor setting and in an outdoor release. In an ambient breeze tunnel test, spores deposited on tiles were reaerosolized and detected by real-time PCR at distances of 30 m from the point of deposition. Real-time PCR signals were inversely correlated with distance from the seeded tiles. An outdoor release of powdered spore simulant at Aberdeen Proving Ground, Edgewood, MD, was monitored from a distance by a light detection and ranging (LIDAR) laser. Over a 2-week period, an array of air sampling units collected samples were analyzed for the presence of viable spores and using barcode-specific real-time PCR assays. Barcoded B. thuringiensis subsp. kurstaki spores were unambiguously identified on the day of the release, and viable material was recovered in a pattern consistent with the cloud track predicted by prevailing winds and by data tracks provided by the LIDAR system. Finally, the real-time PCR assays successfully differentiated barcoded B. thuringiensis subsp. kurstaki spores from wild-type spores under field conditions.

Variability in cell-free expression reactions can impact qualitative genetic circuit characterization.

Cell-free expression systems provide a suite of tools that are used in applications from sensing to biomanufacturing. One of these applications is genetic circuit prototyping, where the lack of cloning is required and a high degree of control over reaction components and conditions enables rapid testing of design candidates. Many studies have shown utility in the approach for characterizing genetic regulation elements, simple genetic circuit motifs, protein variants or metabolic pathways. However, variability in cell-free expression systems is a known challenge, whether between individuals, laboratories, instruments, or batches of materials. While the issue of variability has begun to be quantified and explored, little effort has been put into understanding the implications of this variability. For genetic circuit prototyping, it is unclear when and how significantly variability in reaction activity will impact qualitative assessments of genetic components, e.g. relative activity between promoters. Here, we explore this question by assessing DNA titrations of seven genetic circuits of increasing complexity using reaction conditions that ostensibly follow the same protocol but vary by person, instrument and material batch. Although the raw activities vary widely between the conditions, by normalizing within each circuit across conditions, reasonably consistent qualitative performance emerges for the simpler circuits. For the most complex case involving expression of three proteins, we observe a departure from this qualitative consistency, offering a provisional cautionary line where normal variability may disrupt reliable reuse of prototyping results. Our results also suggest that a previously described closed loop controller circuit may help to mitigate such variability, encouraging further work to design systems that are robust to variability. Graphical Abstract.

Evaluating the persistence and stability of a DNA-barcoded microbial system in a mock home environment.

Recent advancements in engineered microbial systems capable of deployment in complex environments have enabled the creation of unique signatures for environmental forensics operations. These microbial systems must be robust, able to thrive in specific environments of interest and contain molecular signatures, enabling the detection of the community across conditions. Furthermore, these systems must balance biocontainment concerns with the stability and persistence required for environmental forensics. Here we evaluate the stability and persistence of a recently described microbial system composed of germination-deficient Bacillus subtilis and Saccharomyces cerevisiae spores containing nonredundant DNA barcodes in a controlled simulated home environment. These spore-based microbial communities were found to be persistent in the simulated environment across 30-day periods and across multiple surface types. To improve the repeatability and reproducibility in detecting the DNA barcodes, we evaluated several spore lysis and sampling processes paired with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR-associated proteins (Cas) detection (Sherlock). Finally, having optimized the detectability of the spores, we demonstrate that we can detect the spores transferring across multiple material types. Together, we further demonstrate the utility of a recently described microbial forensics system and highlight the importance of independent validation and verification of synthetic biology tools and applications. Graphical Abstract.

The Use and Potential of Biomedical Detection Dogs During a Disease Outbreak.

Biomedical detection dogs offer incredible advantages during disease outbreaks that are presently unmatched by current technologies, however, dogs still face hurdles of implementation due to lack of inter-governmental cooperation and acceptance by the public health community. Here, we refine the definition of a biomedical detection dog, discuss the potential applications, capabilities, and limitations of biomedical detection dogs in disease outbreak scenarios, and the safety measures that must be considered before and during deployment. Finally, we provide recommendations on how to address and overcome the barriers to acceptance of biomedical detection dogs through a dedicated research and development investment in olfactory sciences.

Recombinant protein production in insect larvae: host choice, tissue distribution, and heterologous gene instability.

The expression of the fluorescent protein, DsRed, facilitates the optimization of protein production in orally-infected whole larvae. Trichoplusia ni was shown to be a much better host for recombinant AcMNPV compared to four other noctuid Lepidopteran species achieving 100% infectivity at the minimal tested dose. The highest density of marker protein was found in endothelial and tracheal cells, fat body, and hemocytes. Trichoplusia ni larvae possessed visually detected color over sequential passages of oral infection until the sixth round. Western blot analysis confirmed the progressive decrease of both tetramer and monomer forms of DsRed. The intact DsRed gene and promoter region was present in late passages, but viral population carrying the heterologous gene had dropped more than 2-logs after the fifth round while the amount of total viral DNA remained unchanged over sequential passages.

Risk factors for leg harvest surgical site infections after coronary artery bypass graft surgery.

OBJECTIVE: Harvest site infections are more common than chest surgical infections after coronary artery bypass surgery, yet few studies detail risk factors for these infections. We sought to determine independent risk factors for leg surgical site infections using our institutional Society of Thoracic Surgeons database. METHODS: We retrospectively analyzed data collected from 1980 coronary artery bypass patients undergoing surgery at our institution from January 1, 1996, through June 30, 1999, using The Society of Thoracic Surgeons database. Independent risk factors for leg harvest site infection were identified by multivariate logistic regression. RESULTS: Seventy-six patients (4.5%) were coded as having had a leg harvest site infection, of which 67 were confirmed by infection control. The length of hospital stay after surgery was significantly longer in patients with leg harvest site infection (mean 10.1 days) compared with that of patients without infection (mean 7.1 days, P <.001), and infected patients were more likely to be readmitted to the hospital within 30 days of surgery. Independent risk factors for leg harvest site infection included previous cerebrovascular accident (odds ratio, 2.9), postoperative transfusion of 5 units or more of red blood cells (odds ratio, 2.8), obesity (odds ratio, 2.5), age 75 years or older (odds ratio, 1.9), and female gender (odds ratio, 1.8). CONCLUSIONS: Consistent with previous studies, female gender and obesity were identified as independent risk factors for leg harvest site infection, while previous cerebrovascular accident, postoperative transfusion, and older age are newly described risk factors. The Society of Thoracic Surgeons database is a useful tool for identification of predictors of leg harvest site infections.

The risk factors for deep and superficial chest surgical-site infections after coronary artery bypass graft surgery are different.

OBJECTIVE: We sought to determine risk factors for deep and superficial chest wound infections after coronary artery bypass graft surgery to develop predictive models. METHODS: We retrospectively analyzed data collected on 1980 consecutive patients undergoing coronary artery bypass surgery at our institution between January 1, 1996, and June 30, 1999, by using the Society of Thoracic Surgery database. Independent risk factors for surgical-site infection were identified with multivariate logistic regression. RESULTS: There were 37 (1.9%) deep chest and 46 (2.3%) superficial chest surgical-site infections. Obese diabetic patients had a 7.7-fold increased risk of deep chest infections after controlling for intra-aortic balloon pump use (odds ratio, 3.1) and postoperative transfusion (odds ratio, 2.3). Independent risk factors for superficial surgical-site infections included obesity (odds ratio, 3.1), diabetes in persons 65 years of age or older (odds ratio, 2.7), and current smoking (odds ratio, 2.5). Use of antiplatelet drugs was associated with a lower risk of superficial infections (odds ratio, 0.4). Predicted operative mortality as a marker of severity of illness was not clearly predictive of deep or superficial surgical-site infection. Mortality in the year after the operation was increased in patients with deep chest infections compared with that seen in uninfected control subjects (8/37 [21.6%] vs 114/1612 [7.1%], P =.004) but not in patients with superficial chest infections (7/47 [15.2%] vs 114/1612 [7.1%], P =.075). CONCLUSIONS: Risk factors for deep and superficial chest surgical-site infections after coronary artery bypass surgery differ, suggesting different mechanisms of pathogenesis. Appropriate risk stratification models specific to these important outcomes must be developed.

Structure-based design of supercharged, highly thermoresistant antibodies.

Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.

Intensive care nurses' knowledge of critical care family needs.

AIMS: To explore nurses' knowledge of family needs and to describe their current practices in meeting those needs. BACKGROUND: Accurately assessing and responding to family needs of critically ill patients is significant in reducing the negative impact of stress; strengthens the ability to interact positively; increases family satisfaction with care and promotes trust and confidence. Inadequate attention to complex family needs can result in care fragmentation, family alienation, and the development of adversarial relationships between families and care givers. METHODS: A descriptive correlational quantitative design was utilised for this study, with data collected over a three-month period from nurses working within an Intensive Care Unit. RESULTS: The majority of respondents (n=44) scored above 70% in their knowledge of the needs of family members, indicating an excellent knowledge of those needs but only 4.2% (n=2) were able to rank family needs in order of importance. Whilst nurses reported very good practices in relation to caring for relatives there was no significant statistical relationship found between knowledge scores and self-reported practice indicating that whilst they had the knowledge it is not necessarily translated into clinical practice. But 71.4% (n=34) of respondents claimed their knowledge came from clinical work in ICU and continuing education courses (42%). CONCLUSION: Nurses demonstrated a very good knowledge of the needs of relatives and reported effective nursing interventions in supporting those needs, but knowledge is not necessarily translated into practice.

The persistent problem of new-onset postoperative atrial fibrillation: a single-institution experience over two decades.

OBJECTIVE: Postoperative atrial fibrillation is the most common complication after cardiac surgery. A variety of postoperative atrial fibrillation risk factors have been reported, but study results have been inconsistent or contradictory, particularly in patients with preexisting atrial fibrillation. The incidence of postoperative atrial fibrillation was evaluated in a group of 10,390 patients undergoing cardiac surgery among a comprehensive range of risk factors to identify reliable predictors of postoperative atrial fibrillation. METHODS: This 20-year retrospective study examined the relationship between postoperative atrial fibrillation and demographic factors, preoperative health conditions and medications, operative procedures, and postoperative complications. Multivariate logistic regression models were used to evaluate potential predictors of postoperative atrial fibrillation. RESULTS: Increasing age, mitral valve surgery (odds ratio=1.91), left ventricular aneurysm repair (odds ratio=1.57), aortic valve surgery (odds ratio=1.52), race (Caucasian) (odds ratio=1.51), use of cardioplegia (odds ratio=1.36), use of an intraaortic balloon pump (odds ratio=1.28), previous congestive heart failure (odds ratio=1.28), and hypertension (odds ratio=1.15) were significantly associated with postoperative atrial fibrillation. The non-linear relationship between age and postoperative atrial fibrillation revealed the acceleration of postoperative atrial fibrillation risk in patients aged 55 years or more. In patients undergoing coronary artery bypass grafting, increasing age and previous congestive heart failure were the only factors associated with a higher risk of postoperative atrial fibrillation. There was no trend in incidence of postoperative atrial fibrillation over time. No protective factors against postoperative atrial fibrillation were detected, including commonly prescribed categories of medications. CONCLUSIONS: The persistence of the problem of postoperative atrial fibrillation and the modest predictability using common risk factors suggest that limited progress has been made in understanding its cause and treatment.