Assessment of fungal and bacterial bioaerosols in ambient air in Fairbanks, Alaska

Thesis (M.S.) University of Alaska Fairbanks, 2011Bioaerosols are solid or liquid particles of biological origin that are suspended in the surrounding air or other gaseous environments. Bioaerosols can cause diseases, allergenic or toxicological reactions, respiratory distress, and can be potential bioterrorism threats. Studies concerning ambient bioaerosols have never focused upon central Alaska, and only one experiment has utilized the DRUM (Davis Rotating Unit for Monitoring) impactor as the collection apparatus. This study focuses on the assessment and identification of fungi and bacteria present in the ambient air collected by the DRUM impactor from March 2008 to January 2009. The samples were collected on MylarTM and aluminum substrates (with or without apiezon coating) and subjected to DNA extraction and nested PCR using universal primers for the 16S rRNA gene in bacteria and ITS (internal transcribed spacer) region in fungi. The PCR products were used to generate a clone library, and selected clones from each sample clone library were sequenced. Sequences were taxonomically classified using BLAST for fungal identification and RDP Pipeline for bacterial identification to the genus level. Numerous species of bacteria (i.e., Ralstonia sp., Bradyrhizobium sp., Sphingomonas sp.) and fungi (i.e. Fusarium sp., Cladosporium sp., Penicillium sp.) were identified from the clone libraries, thus indicating that the DRUM impactor has potential for monitoring biological content in the air. The resulting patterns in bacteria and fungi during the course of the year indicate that the DRUM sampler may also have the potential to detect fluctuations in populations that result from meteorological conditions, seasonal cycles, and climatic conditions.Army Research Laboratory Grants W911NF-07-1-0346, W911NF-08-1-0318, W911NF-09-01-05431. Introduction -- 2. Methods & materials -- 2.1. The DRUM impactor -- 2.2. Justification for using DRUM impactor to collect bioaerosols -- 2.3. Bioaerosol collection -- 2.4. Bioaerosol DNA extractions -- 2.5. Bioaerosol DNA amplification and purification -- 2.6. Bioaerosol clone library construction -- 2.7. Taxonomic identification of microbes in bioaerosols -- 2.8. Bioaerosol richness calculations, weather correlations, and PCA analyses -- 3. Results -- 3.1. Community structure of bioaerosols -- 3.2. Microbial diversity of bioaerosols -- 3.3. Microbial communities in relation to weather trends -- 3.4. Monthly microbial composition comparison -- 4. Discussion -- 5. Conclusion -- 6. Acknowledgements -- 7. References -- 8. Appendix

CONSEQUENCE OF CALENDULA OIL ON THE IN- VITRO PERCUTANEOUS ABSORPTION OF DICLOFENAC SODIUM

Terpenes are widely used as penetration enhancers in transdermal drug delivery system. Calendula oil also contains different terpenes.  The enhancing effect of calendula oil on the in- vitro percutaneous absorption of diclofenac sodium (DFS) from carbopol gels containing propylene glycol was investigated. Permeation experiments were performed on artificial skin membrane. The permeation effect of calendula oil on DFS is compared with geraniol and menthol (mild accelerants) . The enhancing effect of calendula oil was found to be more as compared with geraniol and menthol. However, although the addition of calendula oil increased DFS flux; diffusional lag times were longer than for the control ge

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Development, Optimization, and Preclinical Testing of an Impedimetric Aptamer-Based Platinum Wire Biosensing Platform for Cardiac Biomarkers

Cardiovascular diseases (CVDs) are the leading national cause of death, impacting nearly 92.1 million Americans and accounting for 801,000 deaths annually. Unfortunately, CVDs are clinically silent until serious complications arise, thus allowing CVDs to go undetected or even be misdiagnosed at earlier stages. In addition, while biomarker testing and other cardiovascular tests can lead to earlier diagnoses, these tests are usually not ordered unless the probability of the patient having a CVD is high due to the expenses, effort, and time required. Therefore, a rapid point-of-care device would be highly useful for screening CVD conditions. This research effort was designed to fabricate a biosensing platform using aptamers and electrochemical impedance spectroscopy to rapidly detect two of the most prominent CVDs, myocardial infarction (MI) and congestive heart failure (CHF). To detect these two diseases, we screened for corresponding biomarkers Troponin T (TnT) and Brain Natriuretic Peptide (BNP). The first aim focused on fabricating platinum electrode disks using vertically aligned platinum wires cast in epoxy, and optimization of electrode diameter and surface polish. The second aim assessed optimal incubation times, concentrations, and functionalization layer combinations required for sensitive biosensing of biomarkers. The results demonstrated the feasibility of the platform, importance of surface parameters, and significance of each functionalization layer in constructing the biosensor. The third aim focused on testing fully optimized biosensor platforms against rat whole blood samples to assess the impact of (and correct for) factors in whole blood on the biosensor. The corrected biosensor model was tested against clinically derived human serum samples to determine whether the corrected model could accurately detect BNP concentrations. The results demonstrated preliminary efficacy of fabricated biosensor platforms in both serum and whole blood. However, further investigation is required to affirm model accuracy and to miniaturize the platform into a point-of-care device in the future

Causes, epidemiology, and long-term outcome of traumatic cataracts in children in rural India

Purpose : To describe preoperative factors, long-term (>3 years) postoperative outcome and cost of traumatic cataracts in children in predominantly rural districts of western India. Subjects : Eighty-two traumatic cataracts in 81 children in a pediatric ophthalmology department of a tertiary eye-care center. Materials and Methods : Traumatic cataracts operated in 2004-2008 were reexamined prospectively in 2010-2011 using standardized technique. Cause and type of trauma, demographic factors, surgical intervention, complications, and visual acuity was recorded. Statistical Analysis : Data analysis done by using SPSS (Statistical package for social sciences) version 17.0 We have used Chi-square test, Fisher′s exact test, paired t-test to find the association between the final vision and various parameters at 5% level of significance; binary logistic regression was performed for visual outcome ≥6/18 and ≥6/60. Results : The children were examined in a 3-7 year follow-up (4.35 ± 1.54). Average age at time of surgery was 10.4 ± 4.43 years (1.03 to 18). Fifty (61.7%) were boys. Forty (48.8%) were blunt and 32 (39%) were sharp trauma. The most common cause was wooden stick 23 (28.0%) and sharp thorn 14 (17.1%). Delay between trauma and presentation to hospital ranged from same day to 12 years after the injury with median of 4 days. The mean preoperative visual acuity by decimal notation was 0.059 ± 0.073 and mean postoperative visual acuity was 0.483 ± 0.417 (P < 0.001). Thirty-eight (46.3%) had best corrected visual acuity (BCVA) ≥6/18 and 51 (62.2%) had BCVA ≥ 6/60. In univariable analysis, visual outcome (≥6/18) depended on type of surgery (P = 0.002), gender (P = 0.028), and type of injury (P = 0.07)-sharp trauma and open globe injury had poorer outcomes; but not on age of child, preoperative vision, and type of surgeon. On multivariable binary logistic regression, only gender was significant variable. Of the 82 eyes, 18 (22%) needed more than one surgery. The parents spent an average of Rs. 2250 ($45) for the surgery and 55 (66.4%) were from lower socio-economic class. Conclusion : The postoperative visual outcomes varied and less than half achieved ≥ 6/18

Bioactive Synthetic Polymer-Based Polyelectrolyte LbL Coating Assembly on Surface Treated AZ31-Mg Alloys

Polyelectrolyte layer-by-layer (LbL) films on pretreated Mg containing 3 wt.% Al and 1 wt.% Zn (MgAZ31) alloy surfaces were prepared under physiological conditions offering improved bioresponse and corrosive protection. Pretreatments of the model MgAZ31 substrate surfaces were performed by alkaline and fluoride coating methods. The anti-corrosion and cytocompatibility behavior of pretreated substrates were evaluated. The LbL film assembly consisted of an initial layer of polyethyleneimine (PEI), followed by alternate layers of poly (lactic-co-glycolic acid) (PLGA) and poly (allylamine hydrochloride) (PAH), which self-arrange via electrostatic interactions on the pretreated MgAZ31 alloy substrate surface. The physicochemical characterization, surface morphologies, and microstructures of the LbL films were investigated using Fourier-transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The in vitro stability studies related to the LbL coatings confirmed that the surface treatments are imperative to achieve the lasting stability of PLGA/PAH layers. Electrochemical impedance spectroscopy measurements demonstrated that pretreated and LbL multilayered coated substrates enhanced the corrosion resistance of the bare MgAZ31 alloy. Cytocompatibility studies using human mesenchymal stem cells seeded directly over the substrates showed that the pretreated and LbL-generated surfaces were more cytocompatible, displaying reduced cytotoxicity than the bare MgAZ31. The release of bovine serum albumin protein from the LbL films was also studied. The initial data presented cooperatively demonstrate the promise of creating LbL layers on Mg-related bioresorbable scaffolds to obtain improved surface bio-related activity