Optimization of RNA storage in a biobank, as well as methods for manual and automated isolation of RNA from whole blood and leukocyte fraction

Aim. To optimize the technique for the isolation and storage of ribonucleic acid (RNA) from whole blood and leukocyte fraction.Materials and methods. Comparison of isolation quality was carried out for RNA samples obtained from 228 leukocyte samples and 198 whole blood samples. Isolation was performed from fresh and frozen samples using ExtractRNA™ reagent and a MagNA Pure Compact automated system. Various methods of removing erythrocytes (centrifugation and treatment with hemolytic agents from two manufacturers) were tested, as well as freezing with and without preservatives for subsequent RNA isolation.Results. Twenty-one combinations of conditions were tested. The highest quality RNA was isolated by manual extraction using the ExtractRNA™ reagent from a fresh leukocyte fraction, purified by the Amplisens hemolytic agent (successful extraction — 94%, median RIN=8,4); frozen in IntactRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=8); frozen in ExtractRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=9,3).Conclusion. RNA can be isolated from frozen blood fractions, which is not inferior in quality to that isolated from fresh samples. Thus, it is not necessary to isolate RNA immediately after the receipt of biological material.

Childhood Furuncular Cutaneous Myiasis Associated with Seborrheic Dermatitis: A Case Report

Myiasis is a parasitic infection caused by dipterous fly larvae that can affect various organs in both human and animals. Cutaneous myiasis is the most common type of myiasis and can be classified into three categories, localized furuncular myiasis, migratory myiasis, and wound myiasis. One of the risk factors for myiasis is seborrheic dermatitis. The definitive treatment for the condition requires complete extraction of larvae, in combination with oral therapy, and localized occlusion to promote hypoxia. This paper reports a case of a 12-year-old girl with furuncular cutaneous myiasis associated with seborrheic dermatitis on the occipital region that showed significant improvements after manual extraction of larvae after local anesthesia injection of 2% lidocaine on the base of the lesion, in combination with oral and topical therapies

Evaluation of a novel direct capture method for virus concentration n wastewater from COVID-19 infectious ward and correlation analysis with the number of inpatients.

The global outbreak of the SARS-CoV-2 pandemic has increased the focus of Wastewater-based epidemiology (WBE) studies as a tool for understanding the epidemic and risk management. A highly sensitive and rapid method for the virus concentration from wastewater is needed to obtain the accurate information for early detection of SARS-CoV-2 outbreak and epidemic. In this study, we evaluated the efficiency of the direct capture method provided from Promega, based on column adsorption using the wastewater from actual infectious diseases ward. The efficiency of the nucleic acid extraction-purification process was also evaluated by Maxwell RSC instrument (fully automated extraction) and QIAamp Viral RNA mini kit (manual extraction). The obtained SARS-CoV-2 data from wastewater were analyzed with the number of inpatients which is the consideration of the severity and the days of onset. The combination of direct capture and Maxwell's method (DC-MW) was suggested to be a highly sensitive and simple method with better concentration efficiency and quantification than other methods. Moreover, the inpatient conditions (severity and days of after onset) should be considered to accurately understand the actual status of the correlation between the number of inpatients and SARS-CoV-2 concentration in wastewater. The highly sensitive method of DC-MW was suggested to assess more actual situation of SARS-CoV-2 shedding into the wastewater.

Manual extraction of a dislodged and migrated DES from radial artery puncture site

This is a rare case of manual retrieval of a dislodged stent from radial artery access site under local anaesthetic. The LAD PCI was complicated by stent distortion which occurred due to in adequate preparation of a calcified, tortuous lesion. The dislodged stent was unable to be retrieved percutaneously and had to be left in the distal radial artery which a few weeks later migrated from the radial artery and taken out manually from the puncture site.

Identification and field verification of an aggregation pheromone from the white-spotted flower chafer, Protaetia brevitarsis Lewis (Coleoptera: Scarabaeidae)

The white-spotted flower chafer (WSFC), Protaetia brevitarsis Lewis, is native to East Asia. Although their larvae are considered a potential resource insect for degrading plant residues, producing protein fodder, and processing to traditional medicine, adult WSFCs inflict damage to dozens of fruit and economic crops. The control of the WSFC still relies heavily on pesticides and the inefficient manual extraction of adults. Here, we report the identification and evaluation of the aggregation pheromone of WSFCs. From the headspace volatiles emitted from WSFC adults, anisole, 4-methylanisole, 2-heptanone and 2-nonanone were identified as WSFC-specific components. However, only anisole and 4-methylanisole elicited positive dose–response relationship in electroantennography tests, and only 4-methylanisole significantly attracted WSFCs of both sexes in olfactometer bioassays and field experiments. These results concluded that 4-methylanisole is the aggregation pheromone of WSFCs. Furthermore, we developed polyethylene vials as long-term dispensers of 4-methylanisole to attract and kill WSFCs. The polyethylene vial lures could effectively attracted WSFCs for more than four weeks. Pheromone-based lures can be developed as an environmentally friendly protocol for monitoring and controlling WSFC adults.

782. A Comparison of Four Different Sampling Methods Used to Recover Contamination from Environmental Surfaces

Background We hypothesized that sampling tools with the largest surface area would be the most efficient at recovering bacteria. To test this hypothesis, we evaluated four different sampling methods to see which was most effective at recovering bacteria from common environmental surfaces. Methods At UNC Medical Center, a 951 bed academic facility, we tested four collection methods: swabs, RODAC (replicate organism detection and counting) plates, Sponge Sticks with manual extraction and Sponge Sticks using extraction with the Seward Stomacher. Laminate and stainless steel surfaces (100 cm²) were used for testing. Known quantities of Staphylococcus aureus (SA) and Klebsiella pneumoniae (KP) were applied across the test surfaces, then allowed to dry prior to sampling. Swab samples were collected by rubbing a moistened swab over the test surface, followed by a dry swab. Both swab tips were broken into a tube of saline and mixed. An aliquot of each was inoculated to agar plates. Each RODAC agar plate was pressed onto a test surface. Sponge Stick samples were collected by rubbing a pre-moistened sponge over the test surface. Each sponge head was ejected into a bag containing saline. For manual agitation, each bag was kneaded by hand for 1 minute. For Stomacher method, bags were processed for 1 minute. The contents of each bag were poured into tubes, then centrifuged. The supernatant was removed from each tube. An aliquot of each was inoculated to agar plates. Plates were incubated at 35°C, then colonies were counted for each plate. Results Comparative recovery rates across surfaces, organism types and sampling methods are shown in the table. Conclusion Organism type, not sampling method, appeared to the most important factor in bacterial recovery. Recovery of SA was significantly higher than KP, likely because it was able to better withstand manipulation and the physical stress of drying on test surfaces. The sampling tool appeared to have the second largest impact. RODAC yielded the highest recovery, followed by swabs, then sponges. Knowing the variable recovery based on method, surface and organism is important for meaningful interpretation of environmental surveillance conducted during outbreak investigations or quality assurance monitoring. Disclosures William Rutala, MS, MPH, PhD, PDI (Professional Disposibles International) (Consultant) David J. Weber, MD, MPH, PDI (Consultant)

ICESat-2 Marine Bathymetry: Extraction, Refraction Adjustment and Vertical Accuracy as a Function of Depth in Mid-Latitude Temperate Contexts

Nearshore bathymetric data are used in many coastal monitoring applications, but acquisition conditions can be challenging. Shipborne surveys are prone to the risk of grounding in shallow waters, and scheduled airborne surveys often fail to coincide with optimal atmospheric and water conditions. As an alternative, since its launch in 2018, ICESat-2 satellite laser profile altimetry data provide free and readily available data on a 91-day repeat cycle, which may contain incidental bathymetric returns when suitable environmental conditions prevail. In this paper, the vertical accuracy of extracted, refraction-adjusted ICESat-2 nearshore marine bathymetric data is evaluated at four test sites in a Northern hemisphere, temperate latitude location. Multiple ICEsat-2 bathymetric values that occurred in close horizontal proximity to one another were averaged at a spatial scale of 1 m and compared with Multibeam Echosounder bathymetric survey data and Global Navigation Satellite System reference data. Mean absolute errors of less than 0.15 m were observed up to depths of 5 m, with errors of less than 0.24 m (to 6 m), 0.39 m (to 7 m) and 0.52 m (to 10 m). The occurrence of larger bathymetric errors with depth, which increase to 0.54 m at maximum photon depths of 11 m, appears to be primarily related to reduced numbers of geolocated photons with depth. The accuracies achieved up to 6 m suggest that the manual extraction, refraction adjustment and bathymetric filtering steps were effective. Overall, the results suggest that ICESat-2 bathymetric data accuracy may be sufficient to be considered for use in nearshore coastal monitoring applications where shipborne and airborne bathymetric data might otherwise be applied.

The Effect of a Single Freeze–Thaw Cycle on Matrix Metalloproteinases in Different Human Platelet-Rich Plasma Formulations

Storing platelet-rich plasma (PRP) for future use is a compelling approach, presuming the retention of biological properties is maintained. However, certain factors in PRP preparations have deleterious effects for the treatment of certain musculoskeletal conditions. The purpose of this study was to measure and compare matrix metalloproteinase protein (MMP) concentrations between fresh and freeze-thawed leukocyte-rich PRP (LR-PRP) inactivated (LR-I) and activated (LR-A) preparations, and leukocyte-poor PRP (LP-PRP) inactivated (LP-I) and activated (LP-A) preparations. A volume of 60 mL of whole blood was drawn from 19 healthy donors. LP-I and LR-I samples were processed using a manual extraction and centrifugation methodology. LP-A and LR-A products were activated with 10% CaCl2 and recombinant thrombin. Blood fractions were either immediately assayed and analyzed or stored at −80 °C for 24, 72 and 160 h. Multiplex immunoassay was used to measure MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, and MMP-12. MMP-1 concentrations increased in LR-A (p < 0.05) and MMP-9 significantly increased in LR-I (p < 0.05), while MMP-2 significantly decreased in LR-I (p < 0.05) and MMP-3 concentrations significantly decreased in LR-A (p < 0.05). MMP-12 concentrations also significantly decreased in LR-I (p < 0.05) from baseline concentrations. There were no significant differences between LP-A and LP-I preparations and MMP concentrations. MMP-10 concentrations in all PRP samples compared to each freezing time point were also not significantly different. MMPs regulate components of the extracellular matrix (ECM) in the remodeling phase of musculoskeletal injury. In this study, we observed a significant increase and decrease in MMP concentrations in response to a single freeze–thaw cycle in inactivated PRP and activated PRP preparations. This evidence contributes to the growing body of literature on the optimization of PRP preparation and storage strategies prior to delivery. Our findings suggest that specific PRP preparations after a single freeze–thaw may be more advantageous for certain musculoskeletal applications based on the presence of MMP concentrations.

Polish LGBTQ+-Related Anglicisms in a Language Contact Perspective

Research on anglicisms in Polish has nearly a century-long tradition, yet it was Jacek Fisiak’s 1960s–1980s studies on English loanwords that initiated continuous academic interest in anglicisms, coinciding with more intensive English-Polish language contact in post-war Poland. While English loans have been well-researched in the last four decades, the ongoing intensity of English lexical influence on Polish, yielding not only new loans but also new loan types, calls for further studies, especially in the area of quickly developing professional jargons and sociolects. The influx of English-sourced lexis is reflected in the diversity of semantic fields, whose number has grown from 18 (identified in Słownik warszawski 1900–1927 ) to 45 (Mańczak-Wohlfeld 1995). A semantic field that has been underresearched in studies on Polish anglicisms is the LGBTQ+-related lexis, which has drawn from American English gayspeak, shaped by the post-Stonewall gay rights movement initiated in the 1970s. The language data analysed in this study have been collected in a two-stage procedure, which included manual extraction of anglicisms sourced in a diversified corpus of LGBTQ+-related written texts, published in Polish between 2004 and 2020. The second stage involved oral interviews which served a verification function. The aim of this study is to contribute to the lexicographic attempts at researching English-sourced LGBTQ+-related vocabulary in Polish through its identification, excerption, and classification. Assuming an onomasiological approach to borrowing, we arrange LGBTQ+-related anglicisms on a decreasing foreignness scale to identify the borrowing techniques adopted by the recipient language speakers in the loan nativization process. We also address issues related to the identification and semantics of loans, and sketch areas of research on loan pragmatic functions that need further studies.