Prior Extraction Research Articles

Salivary oxytocin after play with parents predicts behavioural problems in preschool children

BackgroundOxytocin (OXT) has attracted research interest for its potential involvement in many of the behavioural problems observed in childhood. Due to its logistical advantages, saliva is an attractive fluid to quantify neuropeptides in children. Salivary OXT has been suggested as a potential biomarker for psychopathology during childhood. However, several questions still remain about the extent to which, and under what conditions, concentrations of OXT in saliva can be reliably measured and are related to behavioural problems in preschool age children. MethodsSeven samples of saliva from 30 preschool children (17 girls) were collected in five different days at their homes. Three of the samples were collected by the children's parents at baseline daily routine conditions, and four of the samples were collected by researchers during two home-visits: before and after two 15-minute dyadic play sessions (one with mothers and one fathers) between each individual parent and the child. Oxytocin concentrations were quantified by Radioimmunoassay with prior extraction. Children's behavioural problems were assessed by the Caregiver-Teacher Report Form (C-TRF) questionnaire, completed by the child's’ preschool teacher. ResultsSalivary OXT measured in baseline samples could not predict any of the behavioural problems measured by the C-TRF. However, when measured after playing with parents salivary OXT showed a stronger pattern of negative correlations, specially with the depression and opposition scales of the C-TRF. Furthermore, salivary OXT was unlikely to be reliably measured using single sampling, but acceptable reliabilities were achieved when averaging several samples. Finally, the single measures of salivary OXT evoked after an episode of play with parents showed better reliabilities than collected at baseline. ConclusionMeasurements of OXT evoked after positive affect interactions with parents seem to capture aspects of the OXT system in young children that might be relevant for understanding the role of this system in children’s social behaviour.

Leadless pacemaker implant in patients requiring CIED extraction: outcomes based upon timing of extraction

Abstract Background Previous results from global Micra Transcatheter Pacemaker clinical trials have demonstrated leadless pacing as a safe and attractive option for patients with prior cardiac implantable electronic device (CIED) infection and extraction. Whether outcomes differ based upon the timing of prior device extraction has not been studied. Purpose To describe characteristics and outcomes of patients undergoing CIED extraction during or prior to Micra implantation. Methods Patients who underwent CIED explant and Micra implantation were identified from the Micra Post-Approval Registry and Micra Acute Performance studies. Baseline characteristics were summarized. A Fine-Gray competing risk model was used to compare risk for major complication through 24 months. Results Of the 2739 patients included in the studies, 99 (3.6%) patients had CIED extraction the day of Micra implantation (same day) and 127 (4.6%) patients had CIED extraction within 30 days prior to Micra implantation (prior). Although infection was the primary reason for CIED extraction in both groups, a larger proportion of prior patients underwent extraction for this reason (87.4% vs. 42.4%). In contrast, more same day patients underwent CIED extraction for physician/elective reasons (16.2% vs. 3.1%). Same day patients prior device history included pacemaker (42 dual chamber and 30 single chamber), ICD (1 single chamber and 4 dual chamber), CRT (7 CRT-ICD and 13 CRT-P) while prior patients device history included pacemaker (29 single chamber, 80 dual chamber), ICD (3 dual chamber), CRT (5 CRT-ICD and 7 CRT-P). Overall, patients with extraction were aged 72.8±14.3 years, predominantly male (65.9%), and medical history was similar between groups, with the exception to CHF, which was higher for the same day group (18.2% vs 6.3%, P=0.021). The implant success rate was 98.0% for same day patients and 100% for prior patients. Median procedure duration was not significantly different between the groups (26.0 minutes and 25.0 minutes for same day and prior, respectively). Average follow-up duration was 16.5±13.8 months (range 0–53.4) for same day patients and 18.2±15.2 months (range 0–58.3) for subsequent patients. The rate of acute major complications (<30 days) was 5.1% for same day and 3.2% for prior. Through 24 months, the rate of major complications was 6.4% for same day and 6.0% for prior (HR: 1.19, 95% CI: 0.40 – 3.50, P=0.76, Figure). The rate of major complications related to infection was low and did not differ by group (1.01% vs. 1.57%, P=1.00) Conclusion The Micra leadless pacemaker was implanted with a high success rate following CIED extraction. Outcomes following CIED extraction appear similar, whether the extraction is performed during or prior to Micra implant. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Medtronic, Inc. Risk of major complications

MP31-02 MICROTESE AND CRYOPRESERVED SPERM - A DECISION-MAKING DILEMMA FOR PATIENTS AND PROVIDERS

You have accessJournal of UrologyInfertility: Therapy (MP31)1 Sep 2021MP31-02 MICROTESE AND CRYOPRESERVED SPERM - A DECISION-MAKING DILEMMA FOR PATIENTS AND PROVIDERS Johnathan Doolittle, Darren Bryk, Scott Lundy, and Sarah Vij Johnathan DoolittleJohnathan Doolittle More articles by this author , Darren BrykDarren Bryk More articles by this author , Scott LundyScott Lundy More articles by this author , and Sarah VijSarah Vij More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002035.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: A known disadvantage of frozen microdissection testicular sperm extraction (mTESE) is the negative impact of cryopreservation and subsequent thawing of any sperm that are obtained. A post thaw (PT) sample provides insight in to how the sample withstands this stress and the predicts likelihood of sperm available for intracytoplasmic sperm injection (ICSI). If a PT cannot be performed due to such low numbers of sperm identified or no pre-freeze motility, providers and couples arrive at a challenging decision in their fertility pathway - pay for an additional sperm retrieval procedure or proceed with a sample that may not be sufficient for ICSI, requiring a back-up plan. Our study sought to characterize the ICSI outcomes after retrieval of motile sperm during frozen mTESE at our institution. METHODS: A retrospective chart review was conducted from 2012 through 2020 to identify all men that underwent frozen mTESE with successful retrieval of motile sperm. Prior sperm extraction or biopsy, testis size, hormone levels, genetics and prior semen analyses were recorded. A PT and recommendations regarding ISCI were made by the lead embryologist at our institution. When available, ISCI outcomes were recorded. RESULTS: Of the 88 men who underwent frozen mTESE, motile sperm were identified in 26.1% (23/88). 16 of these samples were assessed with a PT, of which 12/16 had PT motility. Of these 12, 10 specimens were deemed sufficient to proceed to ISCI by the embryologist whereas the remaining 2 couples were advised to consider fresh mTESE. ICSI records were available for 7 of the 12 patients with PT motility. All 7 cycles led to embryos for transfer, resulting in 6 pregnancies and 5 live births. Of the 11 men without PT motility, ICSI records were available for 6 couples. 3 underwent subsequent fresh mTESE all leading to embryos for transfer but no pregnancies, and 3 proceeded straight to ICSI, 2 of which created an embryo for transfer, with 1 resulting in an ongoing pregnancy. CONCLUSIONS: Of those men who have successful frozen mTESE, as defined as recovery of motile sperm, some may be advised to pursue fresh mTESE based on poor PT parameters or absence of PT due to poor pre-freeze parameters. This creates a very difficult decision for couples. Although frozen mTESE potentially spares the female partner an unnecessary IVF cycle, we often place couples in a difficult position to make a decision about whether to proceed to ICSI absent PT or in the setting of poor PT motility. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e555-e555 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johnathan Doolittle More articles by this author Darren Bryk More articles by this author Scott Lundy More articles by this author Sarah Vij More articles by this author Expand All Advertisement Loading ...

Experimental measurements for the liquid-liquid equilibrium of ternary systems [(water + methyl oleate + model mixtures of tar (naphthalene, phenanthrene and anthracene)] issued from the biomass gasification process

Energy, fuel and chemical production like syngas from biomass is increasingly attracting interest in the world, with increasing potential to replace conventional fossil fuels in the energy market.Biomass gasification has been studied widely as an efficient and sustainable technology for the syngas production.Thus, raw syngas contains a significant quantities of undesirable impurities known as syngas contaminants, especially tar which can cause major problems to the gasification process; therefore it is crucial to eliminate the generated tar in this process by one of the most promising method “the tar scrubbing oil” using a suitable extracting solvent. With this aim, an experimental study of the liquid–liquid equilibrium of the ternary systems of water + solvent (methyl oleate-biodiesel solvent) + model mixtures of tar (naphthalene, phenanthrene and anthracene) that are presented in the raw syngas was conducted at 303.2 K, 323.2 K and 343.2 K. Due to the low solubility of the studied model molecule of tars in the aqueous phase, an extraction of these analytes (naphthalene, phenanthrene and anthracene) from the aqueous phase was performed by an auxiliary solvent (dichloromethane), in which was dissolved an internal standard (hexadecane) before analysis in the GC–MS using the internal standard technique. A control analysis was systematically performed in order to ensure that all of the solute was extracted from the aqueous phase. As for the analysis of the organic phase, it was carried out in the GC–MS using the internal standard technique without any prior extraction. Coulometric Karl Fischer (KF) titration was used to quantify water in the organic phase. Partition coefficient and selectivity of the biodiesel solvent were also discussed. This work is followed by a modeling study of the same systems using the nonrandom two-liquid (NRTL) and UNIQUAC (universal quasichemical) models.

Monitoring Imidazoline Derivatives via Functionalized Nano‐Potentiometric Platforms in Aqueous Humor and Dosage Forms

AbstractThe determination of two imidazoline derivatives [oxymetazoline HCl (OXY) and xylometazoline HCl (XYLO)] was described using different potentiometric platforms. The first electrode type was constructed using tetraphenyl borate (TPB) as anionic exchanger with β‐cyclodextrins (β‐CD) as ionophore forming oxymetazoline‐tetraphenyl borate (OXY‐TPB) and xylometazoline‐tetraphenyl borate (XYLO‐TPB), respectively. The second electrode type was prepared by modification of the first type by conjugation with magnetic iron oxide nanoparticles (MNP) forming (OXY‐MNP) and (XYLO‐MNP). The synthesized electrodes were fully characterized. The effect of magnetic nano‐sized particles as a highly dispersible material with β‐CDs on the electrode characteristics was investigated and compared against the classical electrodes. The response time, working pH range and selectivity coefficients were studied. The functionalized nano‐electrodes (OXY‐MNP and (XYLO‐MNP) were found to be more sensitive than the classical electrodes with linearity ranges (1×10−6–1×10−2 M). The functionalized nano‐electrodes were successfully applied for the in‐line analysis of OXY and XYLO in pharmaceutical dosage forms and spiked rabbit aqueous humor samples with no prior extraction of treatment. This suggests the future use of these electrodes in clinical studies of both drugs of interest.

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

Verification of performance of a direct fluorescent assay for cell-free DNA quantification, stability according to pre-analytical storage conditions, and the effect of freeze-thawing.

A simple fluorescence-based cell-free DNA (CFD) assay has been previously developed that can directly measure nucleic acids without prior DNA extraction and amplification. However, studies on fluorescence-based CFD are lacking. In particular, there is no known information regarding the stability with regard to pre-analytical storage conditions in relation to time and temperature, or on the influence of freeze-thawing. Plasma was directly assayed to measure CFD using PicoGreen™ reagent. Standard linearity and accuracy were confirmed using salmon sperm DNA. Whole blood was left at room temperature (RT) and at 4˚C, and then plasma was separated. The CFD was also measured using thawed plasma after 1 week of freezing. As a correlation with a sperm DNA concentration, CFD demonstrated linearity over a wide range of concentrations, with a 0.998 correlation coefficient. The CFD level showed a change of up to 2.5 µg/ml according to pre-analytical storage time, and the changes were not consistent over time. The CFD values at RT after 1 h were similar to the baseline values, and the relative standard deviation was lowest under this condition. The CFD values between 4˚C and RT were similar over all time periods assessed. After freeze-thawing, the change in CFD value was reduced compared to that before freezing. The present study showed that CFD measurements using plasma processed within 1 h were optimal. Additionally, the effects of substantial changes according to storage conditions were reduced after freeze-thawing, and thus studies using stored samples is viable and relevant.

An improved analytical dynamic model for rotating blade crack: With application to crack detection indicator analysis

Rotating blade is one of the most important components for turbomachinery. Blade crack is one of the most common and dangerous failure modes for rotating blade. Therefore, the fault mechanism and feature extraction of blade crack are vital for the safety assurance of turbomachinery. This study is aimed at the nonlinear dynamic model of rotating blade with transverse crack and the prior feature extraction of blade crack faults based on the vibration responses. First and foremost, a high-fidelity breathing crack model (HFBCM) for rotating blade is proposed on the basis of criterion for stress states at crack section. Since HFBCM is physically deduced from the perspective of energy dissipation and the coupling between centrifugal stress and bending stress is considered, the physical interpretability and the accuracy of the crack model are enhanced comparing with conventional models. The validity of the proposed HFBCM is verified through the comparison study among HFBCM, conventional crack models, and finite element-based contact crack model (FECCM). It is suggested that HFBCM behaves best among the analytical models and matches well with FECCM. With the proposed HFBCM, the nonlinear vibration responses are investigated, and four types of blade crack detection indicators for rotating blade and their quantification method are presented. The numerical study manifests that all these indicators can well characterize the occurrence and severity of crack faults for rotating blade. It is indicated that these indicators can serve as the crack-monitoring indexes.

Deep Learning with ConvNet Predicts Imagery Tasks Through EEG

Deep learning with convolutional neural networks (ConvNets) has dramatically improved the learning capabilities of computer vision applications just through considering raw data without any prior feature extraction. Nowadays, there is a rising curiosity in interpreting and analyzing electroencephalography (EEG) dynamics with ConvNets. Our study focused on ConvNets of different structures, the efficiency of multiple machine learning algorithms with optimization on ConvNets, constructing for predicting imagined left and right movements on a subject-independent basis through raw EEG data. We adapted novel lower-upper triangularization based extreme learning machines (LuELM) to the ConvNet architecture. Results showed that recently advanced methods in machine learning field, i.e. adaptive moments and batch normalization together with dropout strategy, improved ConvNets predicting ability, outperforming that of conventional fully-connected neural networks with widely-used spectral features. The proposed prediction model achieved improvements in classification performances with the rates of 90.33%, 91.00%, and 89.67% for accuracy, recall, and specificity, respectively.

Validation of an UV-Visible spectrophotometry assay method for the determination of chlorpheniramine maleate tablets without prior extraction

Methods proposed by pharmacopeias to check the quality of chlorpheniramine maleate tablets are multi steps methods which involve extraction and present issues with repeatability. An alternative method is proposed with a sensitive, reliable, simple and rapid UV-VISIBLE spectrophotometry method developed for the determination without extraction of chlorpheniramine maleate in tablets. The method was validated using the accuracy profile approach with an accuracy ranging from 99.70 to 100.46%. Analysis was done using 0.25 mol/L sulfuric acid, distilled water, and raw material in a room at 23 °C for 25 to 30 minutes. Chlorpheniramine maleate concentration varied from 0.018 to 0.03 mg/mL. The method was found to be specific with the appearance of the corresponding maxima at 265 nm and a correlation coefficient (R2 ) of 0.9993. Limits of detection and quantification were respectively 1.39 × 10-4and 2.26 × 10-3 mg/mL. The tolerance limits for the different concentration levels (75, 100 and 125%) were respectively: 88.37; 92.74 and91.62% for lower limits and 111.03; 108.17 and 108.02% for upper limits. It was observed that the tolerance limits were within the limits of acceptability set at 20%. Consequently, the method was declared valid and reliable for routine analysis of chlorpheniramine maleate in tablets containing chlorphenamine as active principle.Keywords : Chlorpheniramine maleate, tablets, accuracy profile, assay method without extraction, validation

Insights into OIB-like magmatism contemporaneous with oceanic subduction: Petrogenetic constraints on the Kendelong metagabbro in the North Qaidam

Compared with the statement of the mantle plume hypothesis for large-scale plus high-volume intraplate magmatism, a series of OIB-like mafic rocks which are low-volume and spatially closely related to subduction zones still remain mysterious and inapposite to be explained by using traditional mantle plume hypothesis. In the North Qaidam, most previous studies have focused on ultra-high pressure metamorphic rocks and have paid little attention to mafic magmatism as a mechanism of generating magma with a mantle signature. The Kendelong (KDL) metagabbro with consistently intrusive ages of 485–490 Ma within the Olongbuluke microblock that is overriding plate above the subduction zone is analogous to Oceanic Island Basalts (OIBs) in geochemistry and formed contemporaneously with oceanic subduction. Analyzed samples of the KDL metagabbro have sporadic (87Sr/86Sr)i ratios of 0.7008–0.7063 that have been influenced by paulopost alteration, relatively uniform positive εNd(t) values of +0.3 to +1.4, zircon εHf(t) values of +1.5 to +6.8, and coupled Nd–Hf isotopes that lie on the terrestrial array. After ruling out the possibility of contributions from subducted enriched components and mantle metasomatites, geochemical features and isotopic compositions indicate that the magmas were derived from a relatively primitive asthenosphere without prior large-volume melt extraction. Rare earth element modeling shows that a low degree of partial melting (<10%) of an asthenospheric mantle source in the garnet stability field should be responsible for the magmatic origin of the KDL OIB-like metagabbro. Considering the temporal and spatial correlations between the KDL metagabbro and other types of mafic rock in adjacent regions, we propose that decompression melting of upwelling asthenosphere triggered by rollback of subducted oceanic slab is a plausible regime to explain the petrogenesis of the early Paleozoic KDL OIB-like metagabbro and to trace a fortuitous tectonic transition event. Moreover, even if the OIB-like magmatism is spatially related to subduction zones, the participation of subduction materials could not be an exclusive prerequisite to sentence its genesis.

Application of a Genus-Specific LAMP Assay for Schistosome Species to Detect Schistosoma haematobium x Schistosoma bovis Hybrids.

Schistosomiasis is a disease of great medical and veterinary importance in tropical and subtropical regions caused by different species of parasitic flatworms of the genus Schistosoma. The emergence of natural hybrids of schistosomes indicate the risk of possible infection to humans and their zoonotic potential, specifically for Schistosoma haematobium and S. bovis. Hybrid schistosomes have the potential to replace existing species, generate new resistances, pathologies and extending host ranges. Hybrids may also confuse the serological, molecular and parasitological diagnosis. Currently, LAMP technology based on detection of nucleic acids is used for detection of many agents, including schistosomes. Here, we evaluate our previously developed species-specific LAMP assays for S. haematobium, S. mansoni, S. bovis and also the genus-specific LAMP for the simultaneous detection of several Schistosoma species against both DNA from pure and, for the first time, S. haematobium x S. bovis hybrids. Proper operation was evaluated with DNA from hybrid schistosomes and with human urine samples artificially contaminated with parasites’ DNA. LAMP was performed with and without prior DNA extraction. The genus-specific LAMP properly amplified pure Schistosoma species and different S. haematobium-S. bovis hybrids with different sensitivity. The Schistosoma spp.-LAMP method is potentially adaptable for field diagnosis and disease surveillance in schistosomiasis endemic areas where human infections by schistosome hybrids are increasingly common.

Basalt derived from highly refractory mantle sources during early Izu-Bonin-Mariana arc development

The magmatic character of early subduction zone and arc development is unlike mature systems. Low-Ti-K tholeiitic basalts and boninites dominate the early Izu-Bonin-Mariana (IBM) system. Basalts recovered from the Amami Sankaku Basin (ASB), underlying and located west of the IBM’s oldest remnant arc, erupted at ~49 Ma. This was 3 million years after subduction inception (51-52 Ma) represented by forearc basalt (FAB), at the tipping point between FAB-boninite and typical arc magmatism. We show ASB basalts are low-Ti-K, aluminous spinel-bearing tholeiites, distinct compared to mid-ocean ridge (MOR), backarc basin, island arc or ocean island basalts. Their upper mantle source was hot, reduced, refractory peridotite, indicating prior melt extraction. ASB basalts transferred rapidly from pressures (~0.7-2 GPa) at the plagioclase-spinel peridotite facies boundary to the surface. Vestiges of a polybaric-polythermal mineralogy are preserved in this basalt, and were not obliterated during persistent recharge-mix-tap-fractionate regimes typical of MOR or mature arcs.

Calibration-Less Multi-Coil Compressed Sensing Magnetic Resonance Image Reconstruction Based on OSCAR Regularization.

Over the last decade, the combination of compressed sensing (CS) with acquisition over multiple receiver coils in magnetic resonance imaging (MRI) has allowed the emergence of faster scans while maintaining a good signal-to-noise ratio (SNR). Self-calibrating techniques, such as ESPiRIT, have become the standard approach to estimating the coil sensitivity maps prior to the reconstruction stage. In this work, we proceed differently and introduce a new calibration-less multi-coil CS reconstruction method. Calibration-less techniques no longer require the prior extraction of sensitivity maps to perform multi-coil image reconstruction but usually alternate estimation sensitivity map estimation and image reconstruction. Here, to get rid of the nonconvexity of the latter approach we reconstruct as many MR images as the number of coils. To compensate for the ill-posedness of this inverse problem, we leverage structured sparsity of the multi-coil images in a wavelet transform domain while adapting to variations in SNR across coils owing to the OSCAR (octagonal shrinkage and clustering algorithm for regression) regularization. Coil-specific complex-valued MR images are thus obtained by minimizing a convex but nonsmooth objective function using the proximal primal-dual Condat-Vù algorithm. Comparison and validation on retrospective Cartesian and non-Cartesian studies based on the Brain fastMRI data set demonstrate that the proposed reconstruction method outperforms the state-of-the-art (-ESPIRiT, calibration-less AC-LORAKS and CaLM methods) significantly on magnitude images for the T1 and FLAIR contrasts. Additionally, further validation operated on 8 to 20-fold prospectively accelerated high-resolution ex vivo human brain MRI data collected at 7 Tesla confirms the retrospective results. Overall, OSCAR-based regularization preserves phase information more accurately (both visually and quantitatively) compared to other approaches, an asset that can only be assessed on real prospective experiments.

Evaluation of a commercial microbial enrichment kit used prior DNA extraction to improve the molecular detection of vector-borne pathogens from naturally infected dogs

Accurate detection of vector-borne pathogens (VBPs) is extremely important as the number of reported cases in humans and animals continues to rise in the US and abroad. Validated PCR assays are currently the cornerstone of molecular diagnostics and can achieve excellent analytical sensitivity and specificity. However, the detection of pathogens at low parasitemia still presents a challenge for VBP diagnosis, especially given the very low volume of specimens tested by molecular methods. The objective of this study is to determine if a commercially available microbial enrichment kit, used prior DNA extraction, is capable of expanding the overall microbial community and increasing detectable levels of VBPs in canine blood samples through host DNA depletion. This study used EDTA-whole blood samples from dogs naturally infected with varying parasitemia levels of either Anaplasma phagocytophilum, Babesia gibsoni, or Ehrlichia ewingii. For two VBPs, EDTA-blood samples were diluted to determine the effect of microbial concentration at low parasitemia. Paired EDTA-blood samples from each dog were subjected to traditional, automated DNA extraction with or without the microbial concentrating kit (MolYsis®) prior DNA extraction. Relative amounts of pathogen DNA in paired samples were determined by real-time PCR and Next-Generation Sequencing targeting conserved regions of 16S rRNA (for bacteria) and 18S rRNA (for protozoa). Results from the three molecular methods suggest that the microbial concentrating kit did not improve the detection of VBPs, although significantly reduced the presence of host DNA. Alternative methods for VBP enrichment in clinical samples prior to molecular testing should continue to be investigated, as it may significantly improve clinical sensitivity and reduce the number of false-negative results.

Validation and implementation of a commercial real-time PCR assay for direct detection of Candida auris from surveillance samples.

Rapid and reliable laboratory methods are required for detecting the nosocomial yeast Candida auris. AurisID® (Olm Diagnostics) is a real-time PCR assay approved for detecting C. auris in fungal cultures and directly from blood samples, involving a nucleic acid extraction as a prior step. The purpose of this study is to validate the AurisID® kit for direct detection of C. auris from surveillance samples without prior DNA extraction and to analyse the results of implementing this methodology to our daily laboratory routine protocol for C. auris surveillance studies. Our PCR method using the AurisID® kit was compared with our routine protocol, consisting of culture in CHROMagar® Candida and identification by mass spectrometry. A total of 113 swabs were used for validation and 136 pair of surveillance samples were tested. Limit of detection (LOD) was determined by using swabs in Amies transport medium, which were spiked in a series of dilutions of a C. auris standardised suspension (0.5 McFarland). The PCR method showed high sensitivity, specificity, predictive positive value and predictive negative value (96.6%, 100%, 100% and 98.2%, respectively) when compared with the routine protocol. LOD was 500CFU/ml, which corresponds to approximately 1CFU/PCR. Our PCR method using the AurisID® kit allows a reduction in the turnaround time for surveillance of C. auris compared with other methods. These results are expected to contribute to control C. auris outbreaks, allowing isolation of patients and cleaning of environmental surfaces in advance.

Walnut (Juglans regia L.) Volatile Compounds Indicate Kernel and Oil Oxidation.

Kernel oxidation susceptibility and pellicle darkening are among the biggest concerns regarding walnut quality. Monitoring oxidation is crucial to preserve quality from production to consumption. Chemical oxidation parameters (peroxide value and UV absorbances), fatty acid profile, tocopherols, phenols, and volatiles in ‘Chandler’ and ‘Howard’ kernels were studied at different time points during 28 weeks of storage to evaluate potential oxidation markers. During storage, peroxide value, UV absorbances, and volatiles concentration increased; oxidative stability, phenols, and tocopherols decreased, while fatty acid profile was unaffected. ‘Chandler’ had a lower peroxide value, K232, and K268; and higher kernel and oil oxidative stability compared to ‘Howard’. Phenols and tocopherols decreased 1.2-fold in ‘Chandler’ and 1.3-fold in ‘Howard’. Using multivariate analysis, samples were discriminated in three groups according with their oxidative levels. Increases of volatiles in oil and kernel were associated with higher oxidative levels. Pentanal, 2-methylpropanal, hexanal, (E)-2-pentenal, 3-octanone, octanal, (Z)-2-penten-1-ol, hexanol, (E)-2-octenal, 1-octen-3-ol, benzaldehyde, (E,E)-2,4-nonadienal, and hexanoic acid in kernels were adequate at distinguishing oxidation levels and as oxidative markers in walnuts. Kernel volatiles is a useful measurement for walnut oxidation during storage without any prior fat extraction.

Osteoinduction within adipose tissue fragments by heterodimeric bone morphogenetic Proteins-2/6 and -2/7 versus homodimeric bone morphogenetic protein-2: Therapeutic implications for bone regeneration.

Fragments of subcutaneous adipose tissue that have been genetically modified to express bone morphogenetic protein-2 (BMP-2) regenerate large segmental osseous lesions in rodents. Gene-activated adipose tissue can be implanted into osseous defects without prior cell extraction and cell culture. The present study aimed to explore whether the heterodimers BMP-2/6 or BMP-2/7 exceed the osteoinductive effect of BMP-2 on adipose tissue. In an in vitro tissue culture system, freshly harvested rat subcutaneous adipose tissue was cultivated in the presence of either BMP-2 or BMP-2/6 or BMP-2/7 at a high (200 ng/ml) and low (50 ng/ml) concentration. Gene expression analysis as well as histological and immunohistochemical methods were applied to test for osteoinduction. A concentration of 200 ng/ml of homodimeric BMP-2 induced osteogenic differentiation most potently, showing more calcification and a higher expression level of bone markers than both concentrations of BMP-2/6 or -2/7. A concentration of 50 ng/ml of BMP-2 was a significantly stronger osteogenic inducer than both concentrations of BMP-2/6 and the low concentration of BMP-2/7. The most potent heterodimeric driver of osteoinduction was BMP-2/7 at a high concentration, demonstrating effects similar to those of BMP-2 at a low concentration. Homodimeric BMP-2 evoked osteoinduction within adipose tissue more potently and at a lower concentration than heterodimeric BMP-2/6 or BMP-2/7. This result agrees well with the fact that it might be easier to translate adipose grafts activated by homodimeric BMP-2 clinically. Preclinical in vivo gene transfer studies are necessary to confirm the results of the present study.

Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

Comprehensive utilization of residues of Magnolia officinalis based on fiber characteristics

The traditional Chinese medicinal plant Magnolia officinalis has a wide range of applications; including more than 200 kinds of patented Chinese medicines. Currently well-known application of Magnolia is to extract magnolol from its bark as a bulk drug. However, considerably large amount of residue is generated after the extraction process. Hence, it is crucial to find alternative waste management mechanisms for this biomass resource for the sake of environmental and economic benefits. In this study, Magnolia bark extract residues (MBER) were studied by Energy-dispersive X-ray spectroscopy (EDS), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) analyses for natural fiber synthesis and its potential application for composite material production. The result revealed that prior extraction process has apparently cleaned the fiber surfaces and introduced more –OH groups. Applying the fiber characteristics, addition of 10–25% residue weight contents of MBER as the raw material was carried out, and their corresponding performance using biomass molding technology to prepare trash can products was compared and analyzed. In the end, it was found that the trash cans made by adding 20% MBER had certain performance advantages. Therefore, this study has provided a new use and method for the full utilization of Magnolia officinalis.