Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes.

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application.

A rapid and low-cost method for genomic DNA extraction from the cyanobacterium Synechocystis.

A two-step method is reported for preparation of genomic DNA from the model cyanobacterium Synechocystis that can be performed with minimal equipment and reagents in about an hour. High yields of genetic material can be obtained (200-450 ng/µl) with reasonable purity. A further ethanol precipitation step can be included but is not necessary if template is simply required for polymerase chain reaction (PCR) or digestion. This new protocol is helpful for amplification of genes of interest in early-stage research projects and for low throughput screening of transformants. It is more reliable than colony PCR of Synechocystis cultures, and less involved and cheaper than existing clean-DNA preparation methods. It represents an unusually simple and reliable extraction protocol for the growing body of research making use of this cyanobacterium.

Vacuum ultraviolet absorption spectroscopy in combination with comprehensive two-dimensional gas chromatography for the monitoring of volatile organic compounds in breath gas: A feasibility study.

Vacuum ultraviolet (VUV) absorption spectroscopy was recently introduced as a new detection system for one, as well as comprehensive two-dimensional gas chromatography (GC×GC) and successfully applied to the analysis of various analytes in several matrices. In this study, its suitability for the analysis of breath metabolites was investigated and the impact of a finite volume of the absorption cell and makeup gas pressure was evaluated for volatile analytes in terms of sensitivity and chromatographic resolution. A commercial available VUV absorption spectrometer was coupled to GC×GC and applied to the analysis of highly polar volatile organic compounds (VOCs). Breath gas samples were acquired by needle trap micro extraction (NTME) during a glucose challenge and analysed by the applied technique. Regarding qualitative and quantitative information, the VGA-100 is compatible with common GC×GC detection systems like FID and even TOFMS. Average peak widths of 300ms and LODs in the lower ng range were achieved using GC×GC-VUV. Especially small oxygenated breath metabolites show intense and characteristic absorption patterns in the VUV region. Challenge responsive VOCs could be identified and monitored during a glucose challenge. The new VUV detection technology might especially be of benefit for applications in clinical research.

A Vacuum Ultraviolet Absorption Array Spectrometer as a Selective Detector for Comprehensive Two-Dimensional Gas Chromatography: Concept and First Results.

Fast and selective detectors are very interesting for comprehensive two-dimensional gas chromatography (GC × GC). This is particularly true if the detector system can provide additional spectroscopic information on the compound structure and/or functionality. Other than mass spectrometry (MS), only optical spectroscopic detectors are able to provide selective spectral information. However, until present the application of optical spectroscopy technologies as universal detectors for GC × GC has been restricted mainly due to physical limitations such as insufficient acquisition speed or high detection limits. A recently developed simultaneous-detection spectrometer working in the vacuum ultraviolet (VUV) region of 125-240 nm overcomes these limitations and meets all the criteria of a universal detector for GC × GC. Peak shape and chromatographic resolution is preserved and unique spectral information, complementary to mass spectrometry data, is gained. The power of this detector is quickly recognized as it has the ability to discriminate between isomeric compounds or difficult to separate structurally related isobaric species; thus, it provides additional selectivity. A further promising feature of this detector is the data analysis concept of spectral filtering, which is accomplished by targeting special electronic transitions that allows for a fast screening of GC × GC chromatograms for designated compound classes.

Assessing Caudal Block Concentrations of Bupivacaine With and Without the Addition of Intravenous Fentanyl on Postoperative Outcomes in Pediatric Patients: A Retrospective Review.

Caudal blocks are a significant and efficacious aspect of pediatric anesthesia, especially in urologic and many general surgery cases. This type of regional anesthesia is common because it has a high success rate and provides between 6 and 8 hours of postoperative pain control. The aim of this study was to determine whether the concentration of bupivacaine or the addition of intravascular (i.v.) fentanyl affected the postanesthesia care unit (PACU) discharge time. A retrospective cohort study comparing the outcomes in pediatric patients who have received varying caudal concentrations with and without the addition of i.v. fentanyl was performed. A total of 849 consecutive patients undergoing hypospadias repairs or circumcisions were reviewed and placed in one of the following 3 groups: 0.125% bupivacaine (group 1), 0.25% bupivacaine (group 2), or one of these concentrations of bupivacaine + i.v. fentanyl intraoperatively (group 3). Total PACU time for each group was 46.1 minutes (group 1), 48.9 minutes (group 2), and 49.7 minutes (group 3). Our results revealed that there is no statistically significant difference between concentrations of bupivacaine administered in a caudal block with or without i.v. fentanyl with regard to the outcome of PACU duration (P = 0.16). Overall, based on the retrospective cohort design, there is no difference in primary and secondary outcomes based on the concentration of bupivacaine, when administered at a volume of 1 mL/kg.

Gas chromatography-vacuum ultraviolet spectroscopy for multiclass pesticide identification.

A new vacuum ultraviolet detector for gas chromatography was recently developed and applied to multiclass pesticide identification. VUV detection features full spectral acquisition in a wavelength range of 115-240nm, where virtually all chemical species absorb. VUV absorption spectra of 37 pesticides across different classes were recorded. These pesticides display rich gas phase absorption features across various classes. Even for isomeric compounds, such as hexachlorocyclohexane (HCH) isomers, the VUV absorption spectra are unique and can be easily differentiated. Also demonstrated is the ability to use VUV data analysis software for deconvolution of co-eluting signals. As a universal detector, VUV provides both qualitative and quantitative information. It offers high specificity, sensitivity (pg on-column detection limits), and a fast data acquisition rate, making it a powerful tool for multiclass pesticide screening when combined with gas chromatography.

Vacuum ultraviolet detector for gas chromatography.

Analytical performance characteristics of a new vacuum ultraviolet (VUV) detector for gas chromatography (GC) are reported. GC-VUV was applied to hydrocarbons, fixed gases, polyaromatic hydrocarbons, fatty acids, pesticides, drugs, and estrogens. Applications were chosen to feature the sensitivity and universal detection capabilities of the VUV detector, especially for cases where mass spectrometry performance has been limited. Virtually all chemical species absorb and have unique gas phase absorption cross sections in the approximately 120-240 nm wavelength range monitored. Spectra are presented, along with the ability to use software for deconvolution of overlapping signals. Some comparisons with experimental synchrotron data and computed theoretical spectra show good agreement, although more work is needed on appropriate computational methods to match the simultaneous broadband electronic and vibronic excitation initiated by the deuterium lamp. Quantitative analysis is governed by Beer-Lambert Law relationships. Mass on-column detection limits reported for representatives of different classes of analytes ranged from 15 (benzene) to 246 pg (water). Linear range measured at peak absorption for benzene was 3-4 orders of magnitude. Importantly, where absorption cross sections are known for analytes, the VUV detector is capable of absolute determination (without calibration) of the number of molecules present in the flow cell in the absence of chemical interferences. This study sets the stage for application of GC-VUV technology across a wide breadth of research areas.

Impact of protamine dose on activated clotting time and thromboelastography in infants and small children undergoing cardiopulmonary bypass.

OBJECTIVES: To study the effect of two protamine-dosing strategies on activated clotting time (ACT) and thromboelastography (TEG). BACKGROUND: Protamine dosage based on neutralizing heparin present in the combined estimated blood volumes (EBVs) of the patient and cardiopulmonary bypass (CPB) pump may result in excess protamine and contributes toward a coagulopathy that can be detected by ACT and TEG in pediatric patients. METHODS: A total of 100 pediatric patients 1 month to ≤5 years of age undergoing CPB were included in this retrospective before/after design study. Combined-EBV group consisted of 50 consecutive patients whose protamine dose was calculated to neutralize heparin in the combined EBVs of the patient and the pump. Pt-EBV group consisted of the next 50 consecutive patients whose protamine dose was calculated to neutralize heparin in the patient's EBV. RESULTS: Baseline and postprotamine ACTs were similar between groups. Postprotamine heparin assay (Hepcon) showed the absence of residual heparin in both groups. Postprotamine kaolin-heparinase TEG showed that R was prolonged by 7.5 min in the Combined-EBV group compared with the Pt-EBV group (mean R of 20.17 vs. 12.4 min, respectively, P < 0.001). Increasing doses of protamine were associated with a corresponding, but nonlinear increase in R. There was no significant difference in the changes for K, alpha, and MA between the groups. CONCLUSION: Automated protamine titration with a protamine dosage based on Pt-EBV can adequately neutralize heparin as assessed by ACT while minimizing prolonging clot initiation time as measured by TEG.

Transesophageal Doppler measurement of renal arterial blood flow velocities and indices in children.

BACKGROUND: Doppler-derived renal blood flow indices have been used to assess renal pathologies. However, transesophageal ultrasonography (TEE) has not been previously used to assess these renal variables in pediatric patients. In this study, we (a) assessed whether TEE allows adequate visualization of the renal parenchyma and renal artery, and (b) evaluated the concordance of TEE Doppler-derived renal blood flow measurements/indices compared with a standard transabdominal renal ultrasound (TAU) in children. METHODS: This prospective cohort study enrolled 28 healthy children between the ages of 1 and 17 years without known renal dysfunction who were undergoing atrial septal defect device closure in the cardiac catheterization laboratory. TEE was used to obtain Doppler renal artery blood velocities (peak systolic velocity, end-diastolic velocity, mean diastolic velocity, resistive index, and pulsatility index), and these values were compared with measurements obtained by TAU. Concordance correlation coefficient (CCC) was used to determine clinically significant agreement between the 2 methods. The Bland-Altman plots were used to determine whether these 2 methods agree sufficiently to be used interchangeably. Statistical significance was accepted at P ≤ 0.05. RESULTS: Obtaining 2-dimensional images of kidney parenchyma and Doppler-derived measurements using TEE in children is feasible. There was statistically significant agreement between the 2 methods for all measurements. The CCC between the 2 imaging techniques was 0.91 for the pulsatility index and 0.66 for the resistive index. These coefficients were sensitive to outliers. When the highest and lowest data points were removed from the analysis, the CCC between the 2 imaging techniques was 0.62 for the pulsatility index and 0.50 for the resistive index. The 95% confidence interval (CI) for pulsatility index was 0.35 to 0.98 and for resistive index was 0.21 to 0.89. The Bland-Altman plots indicate good agreement between the 2 methods; for the pulsatility index, the limits of agreement were -0.80 to 0.53. The correlation of the size of the measurement and the mean difference in methods (-0.14; 95% CI = -0.28, 0.01) was not statistically significant (r = 0.31, P = 0.17). For the resistive index, the limits of agreement were -0.22 to 0.12. The correlation of the size of the measurement and the mean difference in methods (-0.05; 95% CI = -0.09, -0.01) was not statistically significant (r = 0.10, P = 0.65). CONCLUSION: This study confirms the feasibility of obtaining 2-dimensional images of kidney parenchyma and Doppler-derived measurements using TEE in children. Angle-independent TEE Doppler-derived indices show significant concordance with those derived by TAU. Further studies are required to assess whether this correlation holds true in the presence of renal pathology. This technique has the potential to help modulate intraoperative interventions based on their impact on renal variables and may prove helpful in the perioperative period for children at risk of acute kidney injury.

Mitomycin C in the treatment of a Schneiderian (inverted) papilloma of the lacrimal sac.

Schneiderian papillomas are benign tumours, with an aggressive nature. A case with a recurrent Schneiderian papilloma of the nasolacrimal sac, who underwent an external dacryocystorhinostomy (DCR) with irrigation of the nasolacrimal system with 0.02% mitomycin C (MMC), is presented at 18 months follow-up. External DCR using MMC, as an adjuvant therapy, is a novel approach to the treatment of Schneiderian papilloma of the nasolacrimal tract. It allows preservation of function, compared with the conventional treatment of dacryocystectomy.

A comparison of laser-assisted drug delivery at two output energies for enhancing the delivery of topically applied LMX-4 cream prior to venipuncture.

BACKGROUND: Laser-assisted drug delivery (LAD) has the potential for facilitating topical anesthesia with reduced onset time. METHODS: In this randomized, double-blind, crossover study we compared the efficacy and adverse event profile of LAD for topical anesthesia before venipuncture using two output energies (2.0 and 3.5 J/cm2). RESULTS: Mean Visual Analog Scale pain scores were not statistically different (P = 0.57) between the low-energy (mean = 6.7) and high-energy (mean = 8.1) lasers. CONCLUSIONS: LAD at an energy of 2.0 J/cm2 (570 mJ) is as effective, with similar adverse events, as an energy of 3.5 J/cm2 (1000 mJ) in facilitating topical anesthesia.

Effect of disease-related pain on the health-related quality of life of children and adolescents with cystic fibrosis.

OBJECTIVES: The authors' objective was to describe the effect of recurrent pain symptoms on the health-related quality of life (HRQOL) of children and adolescents with cystic fibrosis (CF). Frequent pain, independent of disease severity, was hypothesized to be associated with broad decrements in physical, psychological, and social functioning. METHODS: Forty-six children and adolescents (mean age 12.9 years; 52% female) completed the Cystic Fibrosis Questionnaire-Revised (CFQ-R) to assess their HRQOL and a retrospective pain interview to assess pain location, frequency, intensity, duration, and bother during a routine clinic visit. Forced expiratory volume in 1 second as percent of predicted (FEV1%) was recorded from medical charts to assess the children's disease severity. RESULTS: As hypothesized, pain symptoms were related to children's physical, emotional, and role functioning, CF-related symptoms, and overall perception of their health. Children with frequent pain had significantly reduced physical functioning, vitality, role limitations, and overall perceptions of their health, and increased eating disturbances, treatment burden, respiratory, and digestive symptoms compared with children with no pain or less frequent pain. After controlling for the effects of disease severity, pain frequency continued to predict children's HRQOL in each of these areas. DISCUSSION: Children with frequent CF-related pain experienced broad decrements in their HRQOL. These findings are similar to those found in other populations of children experiencing disease-related pain, suggesting a pervasive impact of pain on overall health and well-being. Future research is needed to evaluate treatments to reduce pain symptoms and improve HRQOL in children with cystic fibrosis.

Assessment of acute and chronic pain symptoms in children with cystic fibrosis.

Pain is important to assess and treat in children with cystic fibrosis (CF), because pain symptoms may limit children's ability to participate in their CF-related care and may reduce their overall well-being and quality of life. The aims of this study were to: 1) assess acute and chronic pain symptoms as reported by children with CF, and 2) examine the relationship between pain symptoms and disease severity as measured by percentage of forced expired volume in 1 sec (FEV1%). Forty-six children completed a self-report questionnaire assessing characteristics of chronic disease-related pain (frequency, intensity, duration, associated emotional upset, and location of pain). Children also rated their pain intensity associated with common CF-related procedures. The primary locations of pain reported were the abdominal/pelvic region, chest, and head/neck. Forty-six percent of the sample described pain occurring at least once per week. Most children reported their pain intensity as mild and of short duration. However, a small subgroup of children reported longer-lasting and moderately intense pain. Children with chest pain were found to be particularly at risk for experiencing more functional limitations and a significantly lower FEV1% compared to children without chest pain. The majority of children reported that nonpharmacological therapies (e.g., medication, rest, or distracting activities) provided some pain relief. Disease-related pain is common for children and adolescents with CF, suggesting that pain assessment should be a routine part of their clinical care. Further research is clearly needed to better understand the sources of pain and how best to provide relief.

A randomized, double-blind comparison study of EMLA and ELA-Max for topical anesthesia in children undergoing intravenous insertion.

BACKGROUND: Topical anesthetics may help reduce discomfort associated with procedures involving needle-puncture, such as intravenous (i.v.) insertions, in children. EMLA cream has become a common, noninvasive therapy for topical anesthesia in children. ELA-Max is a recently introduced topical anesthetic cream marketed as being as effective in producing topical anesthesia after a 30-min application as EMLA is after a 60-min application. The purpose of this research was to compare ELA-Max at 30 min with EMLA at 60 min for providing topical anesthesia for i.v. insertions in children. METHODS: Sixty children, ages 8-17 years, requiring an i.v. were randomized to receive either the 30 min application of ELA-Max (n = 30) or the 60 min application of EMLA (n = 30). Children rated any pain associated with the i.v. insertion using a 100-mm Visual Analog Scale (VAS). The anesthesiologist assessed the presence of blanching at the site and rated the difficulty of placing the i.v. RESULTS: There was no clinically or statistically significant difference in pain ratings (P = 0.87) between the ELA-Max (mean = 25.7) and the EMLA (mean = 26.8) groups. ELA-Max caused significantly (P = 0.04) less blanching than EMLA, however there was no difference in the anesthesiologists' rating of the difficulty of the i.v. placement between the groups (P = 0.73). CONCLUSION: Results from this study support the claim that a 30-min application of ELA-Max (with occlusion) is as effective as a 60-min application of EMLA (with occlusion) for producing topical anesthesia for i.v. insertion in children.

Preoperative neodymium:YAG anterior capsulotomy in intumescent cataract: preventing extension of the capsular tear to the lens periphery.

Extension of the capsulotomy into the periphery is a well-known complication of cataract surgery in young patients with intumescent cataracts. We report such a case successfully treated preoperatively with a neodymium:YAG laser anterior capsulotomy.