Laminar Chamber Research Articles

An Assessment of Surface Contamination and Dermal Exposure to 5-Fluorouracil in Healthcare Settings by UPLC-MS/MS Using a New Atmospheric Pressure Ionization Source

5-Fluorouracil (5-FU) is a well-known cytostatic drug, which is often used in cancer treatments. Yet, it is also a very dangerous compound for people who are occupationally exposed to it for a long time, such as pharmacy employees, nurses and cleaning staff. We aimed to improve and implement a LC-MS/MS method for 5-FU quantification on surface contamination samples collected with swabs in a pharmacy department and outpatient nursing station of a university hospital. To improve the existing methods to quantify 5-FU, we compared a LC-MS/MS method using the frequently applied electrospray ionization source (ESI) with a UniSpray ionization source (USI). To determine the contamination of 5-FU in a pharmacy department preparing 5-FU infusion bags, which are then given to patients in the outpatient nursing stations, we collected multiple surface swabs of the laminar flow cabinets and frequently touched objects, before the preparation and administration of 5-FU and afterwards. Furthermore, we sampled the protective gloves and the bare hands of employees of the pharmacy department, involved in the preparation of the infusion bags. Using the USI source, we were able to reach the lowest limit of quantification (LOQ). With this technique, we were able to detect 5-FU contamination on the laminar flow cabinets and frequently used objects in the pharmacy department and the outpatient nursing station in the very low ng/cm2 range. This contamination was mostly higher after preparation or administration than before. While we also found 5-FU on the protective gloves, we almost found no 5-FU on the skin of the pharmacy technicians preparing the 5-FU infusion bags. In conclusion, our method was able to detect very low concentrations of 5-FU contamination, but the contamination we found is very unlikely to result in any issues for the personnel working in these areas.

Evaluation of the Physical Compatibility of Intravenous Methocarbamol in Lactated Ringer's, 0.45% Normal Saline, and Plasma-Lyte A.

Background: The need to determine physical compatibility of intravenous admixtures is directly related to patient safety and patient outcomes. While the provision of multi-modal analgesic strategies has increased over the past decade, a paucity of data exists regarding physical compatibility of select medications. Objectives: To evaluate the physical compatibility of methocarbamol in Lactated Ringer's (LR), 0.45% normal saline (0.45% NaCl), and Plasma-Lyte A (PLA) at concentrations of 4, 10, and 20 mg/mL. Methods: Admixtures were prepared and evaluated using previously validated methods under a laminar flow hood using aseptic technique. Samples were prepared in a triplicate manner, 3 mL aliquots were placed into polymethyl methacrylate cuvettes for evaluation at time points 0, 1, 5, 8, and 24 hours. Visual inspection of samples included assignment of a number: 0-no precipitation, 1-trace evidence of precipitation, 2-slight haze, 3-medium haze, and 4-heavy precipitation. Any evidence of precipitation was considered significant. A variable wavelength spectrophotometer set at 547 nanometers was used to measure absorbance. A change in absorbance of ±0.010 was considered significant. A change in pH of ±0.1 was considered significant. Results: No significant changes occurred relating to visual inspection or absorbance across all concentrations and time points for LR; however, there was a significant change in pH across all concentrations at hour 5. In 0.45% NaCl and PLA, no remarkable changes occurred across all concentrations and time points regarding visual observation, spectrophotometric absorbance, and pH analysis. Conclusions: Methocarbamol at concentrations of 4, 10, and 20 mg/mL is physically compatible for up to 1 hour in LR. Methocarbamol is physically compatible in 0.45% NaCl and PLA for up to 24 hours. Chemical stability tests are warranted to confirm these findings.

THE EFFECTS OF DIFFERENT SONICATION METHODS ON ALPHA-SYNUCLEIN PRE-FORMED FIBRILS

Alpha-synuclein (α-syn) aggregation is associated with neuronal death and the pathological hallmark of Parkinson's disease (PD). The α-syn preformed fibril model (α-syn-PFFs), reflects α-syn aggregation and is currently used in PD studies. To pass through the cell membrane, long fibrils should be fragmented by sonication. In our study, the effects of temperature, pulse modifications and/or device type on the sonication of α-syn-PFFs were investigated. Sonication was performed ultrasonic bath and in laminar-flow cabinet with probe sonicator. Dilutions were made from 5 µg/µl α-syn-PFFs stock in sterile-filtered dH2O to a final concentration and volume of 0.1 µg/µl and 200µl, respectively. Sonication was performed in an ultrasonic bath containing water at 10°C for 1 hour. All probe sonications were performed at 30% amplitude for 1 minute and 20 repetitions. The effect of temperature on sonication has been evaluated by performing sonication at room temperature (RT), in ice and in ice surrounded by dry ice. Also, the effects of pulse duration on sonication were evaluated using pulse durations of 1second(sec) on/1sec off, 3sec on/3sec off and 5sec on/5sec off. Furthermore, by waiting one minute between each sonication cycle, the heat released by the probe was prevented from affecting the fibrillar structure. The particle size was measured in triplicate by dynamic light scattering method. For transmission electron microscopy, formvar/carbon-coated grids were run through ddH2O-sonicated fibril-uranyl acetate solutions and kept dry until examined. Due to the variation in breakage of long α-syn fibrils, the effect of different parameters on sonication was investigated. In comparison of pulse durations, 5sec on/5sec off application produced shorter fibrils. Comparing the temperature interventions, lowering the temperature decreased the fibril size at 1sec on/1sec off settings but increased it at 3sec on/3sec off and 5sec on/5sec off. However, the shortest fibrils were obtained by sonication for 5sec on/5sec off at RT

First Report of Xanthomonas euvesicatoria pv. sesami Causing Leaf Spot on Sesame (Sesamum indicum L.) in South Carolina, USA.

Sesame (Sesamum indicum L.) is an annual plant known as one of the first domesticated oilseed crops. It is cultivated worldwide, mostly in Asia, Africa, and the Americas (Singh, 2006). In August 2022 and September 2023, dark angular necrotic spots on leaves and stems (100% incidence), blights, and severe defoliation were observed in a 4-acre rainfed sesame field located in the Colleton County of South Carolina, USA (Fig. S1). Bacterial streaming from cut leaf lesions was observed from diseased plants in both years. Two plants were collected for pathogen isolation in 2023. Symptomatic leaves were surface sterilized with 70% ethanol for 1 min and dried in a laminar flow hood. For each isolate, four sterile toothpicks were used to poke lesion margins and stirred in 300 µl of sterile distilled water in a 2-ml sterile microcentrifuge tube and soaked at room temperature (c. 21 °C) for 10 min. Each bacterial suspension (10 µl) was streaked on nutrient agar (NA) in a Petri dish. Convex and mucoid yellow colonies formed after a 48-h incubation at 28°C in the dark. Two isolates (S813 and S814), one from each plant, were obtained by transferring single colonies to new NA plates. Both isolates were preliminarily identified as Xanthomonas [S813: X. campestris (P = 0.53); S814: X. campestris (P = 0.77)] using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of the atpD and dnaK genes was performed for both isolates using the conditions described in Félix-Gastélum et al. (2019). The sequences of both amplicons are 100% identical for each gene between the two isolates. PCR and sequencing of the gyrB gene was also done for S813 with the primers from Young et al. (2008). The atpD (S813/S814), dnaK (S813/S814), and gyrB (S813) sequences (GenBank accessions: PP507118 to PP507120) showed the best match with 100% identity to the corresponding gene sequences [GenBank accessions: KJ491167 (100% coverage), KJ491257 (99% coverage), EU285201 (100% coverage)] of the X. euvesicatoria pv. sesami (=X. campestris pv. sesami) type strain LMG865 (Constantin et al. 2015, Parkinson et al. 2009). A neighbor joining tree with the concatenated sequences of these three genes (2,210 nt) showed that S813 and LMG 865 had the closet relationship with X. euvesicatoria pv. alfalfae (CFBP3836, Fig. S2). To fulfill Koch's postulates, three healthy sesame plants (cultivar Shirogoma) were spray inoculated separately with each suspension of S813 and S814 in sterile tap water until runoff (approx. 5×108 CFU/ml). Two sesame plants were sprayed with sterile tap water and served as negative control. All plants were maintained in a greenhouse at approximately 28/20°C (day/night) with natural photoperiod. Dark leaf spots and leaf yellowing were observed on inoculated plants 7 to 14 days after inoculation. No disease symptom was observed on the control plants. Bacteria were reisolated from leaf spots of the inoculated plants and confirmed to be X. euvesicatoria pv. sesami based on atpD and dnaK sequences. The disease was first reported in Sudan (Sabet and Dowson, 1960), after which it was reported in USA (Isakeit et al., 2012) and Mexico (Félix-Gastélum et al. 2019). To the best of our knowledge, this is the first report of this disease in South Carolina, USA. Since the interest of sesame to the farmers is increasing in the southeastern USA, it is necessary to perform further research to examine the disease distribution and its economic impact.

Design and Construction of a Radiochemistry Laboratory and cGMP-Compliant Radiopharmacy Facility.

The establishment of a compliant radiopharmacy facility within a university setting is crucial for supporting fundamental and preclinical studies, as well as for the production of high-quality radiopharmaceuticals for clinical testing in human protocols as part of Investigational New Drug (IND) applications that are reviewed and approved by the U.S. Food and Drug Administration (FDA). This manuscript details the design and construction of a 550 ft2 facility, which included a radiopharmacy and a radiochemistry laboratory, to support radiopharmaceutical development research and facilitate translational research projects. The facility was designed to meet FDA guidelines for the production of aseptic radiopharmaceuticals in accordance with current good manufacturing practice (cGMP). A modular hard-panel cleanroom was constructed to meet manufacturing classifications set by the International Organization of Standardization (ISO), complete with a gowning room and an anteroom. Two lead-shielded hot cells and two dual-mini hot cells, connected via underground trenches containing shielded conduits, were installed to optimize radioactive material transfer while minimizing personnel radiation exposure. Concrete blocks and lead bricks provided sufficient and cost-effective radiation shielding for the trenches. Air quality was controlled using pre-filters and high-efficiency particulate air (HEPA) filters to meet cleanroom ISO7 (Class 10,000) standards. A laminar-flow biosafety cabinet was installed in the cleanroom for preparation of sterile dose vials. Noteworthy was a laminar-flow insert in the hot cell that provided a shielded laminar-flow sterile environment meeting ISO5 (class 100) standards. The design included the constant control and monitoring of differential air pressures across the cleanroom, anteroom, gowning room, and controlled research space, as well as maintenance of temperature and humidity. The facility was equipped with state-of-the-art equipment for quality control and release testing of radiopharmaceuticals. Administrative controls and standard operating procedures (SOPs) were established to ensure compliance with manufacturing standards and regulatory requirements. Overall, the design and construction of this radiopharmacy facility exemplified a commitment to advancing fundamental, translational, and clinical applications of radiopharmaceutical research within an academic environment.

Advanced optical photothermal infrared spectroscopy for comprehensive characterization of microplastics from intravenous fluid delivery systems

Growing attention is being directed towards exploring the potential harmful effects of microplastic (MP) particles on human health. Previous reports on human exposure to MPs have primarily focused on inhalation, ingestion, transdermal routes, and, potentially, transplacental transfer. The intravenous transfer of MP particles in routine healthcare settings has received limited exploration in existing literature. Standard hospital IV system set up with 0.9 % NaCl in a laminar flow hood with MP contamination precautions. Various volumes of 0.9 % NaCl passed through the system, some with a volumetric pump. Fluid filtered with Anodisc filters washed with isopropyl alcohol. The IV cannula was immersed in Mili-Q water for 72 h to simulate vein conditions. Subsequently, the water was filtered and washed. Optical photothermal infrared (O-PTIR) microspectroscopy is used to examine filters for MP particles. All filters examined from the IV infusion system contained MP particles, including MPs from the polymer materials used in the manufacture of the IV delivery systems (polydimethylsiloxane, polypropylene, polystyrene, and polyvinyl chloride) and MP particles arising from plastic resin additives (epoxy resin, polyamide resin, and polysiloxane-containing MPs). The geometric mean from the extrapolated result data indicated that approximately 0.90 MP particles per mL of 0.9 % NaCl solution can be administered through a conventional IV infusion system in the absence of a volumetric pump. However, with the implementation of a pump, this value may increase to 1.57 particles per mL. Notably, over 72 h, a single cannula was found to release approximately 558 MP particles including polydimethylsiloxane, polysiloxane-containing MPs, polyamide resin, and epoxy resin. Routine IV infusion systems release microplastics. MP particles are also released around IV cannulas, suggesting transfer into the circulatory system during standard IV procedures.

Cryopreservation of sessile oak (Quercus petraea (Matt.) Liebl.) plumules using aluminium cryo-plates: influence of cryoprotection and drying

Background Quercusseeds that are recalcitrant to desiccation and freezing temperatures cannot be stored in gene banks under conventional conditions. However, the germplasm of some recalcitrant seeded species can be stored in liquid nitrogen (–196 °C). Unfortunately, for many species, among them for almost the whole genus Quercus, an effective cryostorage method is still unknown. In this study, we propose a successful cryostorage protocol for Quercus petraea (Matt.) Liebl. germplasm using plumules (a shoot apical meristem of an embryo) frozen on aluminium cryo-plates.ResultsThe plumules isolated from the acorns of ten provenances were prestored in 0.5 M sucrose solution (for 18 h). To form alginate beads (one plumule per bead), the plumules were placed in the wells of a cryo-plate and embedded in calcium alginate gel. For cryoprotection, the encapsulated plumules were immersed in cryoprotectant solution containing 2.0 M glycerol and different concentrations of sucrose (0.8–1.2 M) for 40 min at 25 °C and desiccated under a laminar flow cabinet for 1.0–4.0 h. Cryo-plates with plumules were directly immersed in liquid nitrogen and then cryostored for 30 min. For rewarming, cryo-plates with plumules were immersed in 1.0 M sucrose solution and rehydrated for 15 min at 25 °C. Survival rates varied from 25.8 to 83.4 were achieved after cryoprotection in 1.0 M sucrose solution and the drying of plumules for 2 h. The in vitro regrowth rate of cryopreserved plumules varied among provenances and was 26–77%.ConclusionsThis study presents, for the first time, a successful, simple and effective protocol for the cryopreservation of Q. petraea germplasm that could be used in gene banks. The experiment was successfully repeated on seeds from various provenances, each yielding similar, good results. However, seed quality and storage time after harvesting are important factors in plumule regrowth after cryopreservation.

IMPACT OF A GEL COMPOSITION CONTAINING VARIOUS SUBSTANCES ON INDICES OF IMMUNITY AND TISSUE INFLAMMATION IN PALATINE TONSILS OF PATIENTS WITH CATARRHAL GINGIVITIS DURING CHRONIC TONSILLITIS

The study of pharmacotherapeutic (immunomodulating) action in conditions of in vitro cell cultures is currently considered appropriate and justified for determining the action of drugs at the preclinical level. It is also important to address the pathogenetic nature in using such agents, especially fro local application. This study investigated the efficacy of a multicomponent gel containing decamethoxine and hyaluronic acid in treating chronic catarrhal gingivitis among patients with chronic tonsillitis. The purpose of the work is to assess the efficacy of a gel containing decamethoxine and hyaluronic acid on the tissue of the palatine tonsils in the therapy of chronic catarrhal gingivitis in patients with chronic tonsillitis.
 Materials and methods. To investigate the effects of the main active components in the gel, we followed the recommendations of I.P. Kaidashev et al. for testing pharmacological preparations in in vitro cultures. Tissue samples from the palatine tonsils were obtained from 20 patients with chronic tonsillitis and chronic catarrhal gingivitis undergoing tonsillectomy. All procedures adhered to the guidelines set by the Laboratory of Pathophysiology and Immunology at Prof. O.S. Kolomiichenko Institute of Otolaryngology, National Academy of Sciences of Ukraine. Palatine tonsil tissue was incubated in 199 medium (Serva, Germany) containing gentamicin sulfate (Darnytsa, Ukraine) at a concentration of 120 μg/ml for 20 minutes at 4-8°C. After that, the hemorrhagic parts were removed from the whole tonsil and placed into Eagle's medium (Sigma, USA) and rinsed three to four times. Subsequently, the prepared tissue was placed in sterile vials containing enriched Eagle's medium supplemented with L-glutamine, sodium bicarbonate, 100x vitamin concentrate, 5% fetal calf serum, and 60 μg/mL gentamicin. Tonsil tissue was dissected using scissors and then washed repeatedly (5-7 times) with fresh medium to remove loosely attached cells, then, the resulting cell suspension was filtered. All procedures were conducted within a sterile laminar flow cabinet. Cell viability was assessed using light microscopy (Olympus-23, Japan) and a hemocytometer. A final cell suspension containing 2.5 million lymphocytes per milliliter of medium was prepared. Suspensions with a viability of less than 90% (blue, "dead" cells) were excluded from further experiments.
 Results. Treatment of in vitro palatine tonsil cells from patients with chronic catarrhal gingivitis and recurrent tonsillitis with the main components of the developed gel (decamethoxine and hyaluronic acid) did not induce significant changes in the concentration of α and γ-interferons. Our findings suggest that the gel composition reduces the levels of pro-inflammatory factors: interleukin-1β and immune complexes. Additionally, it appears to stimulate the production of the antimicrobial factor antistreptolysin-O by tonsil cells, potentially leading to increased antibody production against hemolytic streptococcus antigens.
 Conclusion. Based on the in vitro findings, the investigated gel composition containing decamethoxine and hyaluronic acid demonstrates potential anti-inflammatory and antimicrobial properties that stimulate the production of the antimicrobial factor antistreptolysin-O by tonsil cells, which might lead to increased antibody production against hemolytic streptococcus antigens.

First report of Calonectria ilicicola causing red crown rot of soybean in Indiana.

Soybean (Glycine max [L.] Merr.) samples from commercial fields in Decatur and Spencer counties, Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab in August to October 2022. Plants exhibited whole-leaf to interveinal chlorosis of the foliage, red to dark brown external lesions on the crown spreading from the soil-line upward, and severe root rot. In the fields, patches of diseased plants were observed, with greater than 50% of the plants affected and yield loss up to 50%. Orange to red perithecia were present on the exterior of symptomatic stem tissue and ranged in size from 329 to 433 × 232 to 306 µm (n = 10). Stems were surface sterilized in 10% Clorox (0.825% NaOCl) for 1 min, then rinsed with sterile distilled water and dried. In a laminar flow hood, sections of symptomatic stem tissue were plated on using quarter-strength potato dextrose agar (QPDA) and incubated under fluorescent lights on a 12-hr light/dark cycle at 20°C. After 6 days, fungal colonies with fluffy aerial hyphae, which were white near the colony margins and orange to burnt-red near their center, grew uniformly from the stem tissue plated. Elongate, cylindrical hyaline conidia with zero to three septations measuring 45.5 to 73.8 × 4.4 to 6.7 µm (n = 22) grew in clusters from symptomatic stem tissue within the plate. Perithecia developed after 14 days. Falcate, hyaline ascospores with one to two septa measuring 29.4 to 54.7 × 4.6 to 6.8 (n = 23) µm developed within the perithecia. Calonectria ilicicola Boedijn & Reitsma was confirmed based on morphological characteristics (Padgett et al. 2015). Isolate PPDL 22-01457B was used for DNA extraction using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research, Irvine, CA). The internal transcriber region (ITS), actin (ACT) and β-tubulin (TUB2) genes were amplified (Carbone and Kohn 1999; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; White et al. 1990). Amplicons were sent for Sanger sequencing (Genewiz, Inc., South Plainfield, NJ), submitted to Genbank, and assigned accession numbers ITS: OQ932995, Actin: OR484986, and β-tubulin: OR546281. Sequences were analyzed using the NCBI BLASTn tool with results showing 99.5 to 100% identical to C. ilicicola (GenBank accessions LC500063, OQ303403, CP085825, respectively). To perform Koch's Postulates, 90 soybean seeds (CP3620E) were planted in potting media (Berger, Saint-Modeste, Quebec, Canada) in a seed flat with 45 of the plants used as controls and grown under grow lights for 16hr light/8hr dark at 20℃. Individual seedling crowns were inoculated 3 days post-emergence with a 5 to 10 ml spore and hyphal suspension that was scraped from the surface of a 14-day old QPDA culture after adding 300 mL deionized (DI) to each plate grown at 20 to 22°C. The control plants received sterile-DI water. Plants were covered in a plastic bag for 72 h. Plant stems were sprayed with sterile-DI water once a day for seven days. Symptoms were observed after four days, but significant crown rot and lesions developed after two weeks before wilting and dying. Calonectria iliciola was isolated uniformly from symptomatic plants and identified morphologically. Control plants showed no symptoms. Inoculations were repeated 3 times with similar results. As of fall 2023, red crown rot has been confirmed in Adams and Rush counties in Indiana. Red crown rot has been confirmed in several Midwest states (Kleczewski et al. 2019, Neves et al. 2023), but the extent of its distribution and disease management strategies are still limited.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

First report of root and crown rot caused by Fusarium oxysporum Schltdl. on Prunus cerasifera L. in Spain.

In 2021, Spain was the largest producer of cherries in Europe with a production of 125810 tons (FAOSTAT, 2021). In May 2022, in several production fields in Huelva (Spain), wilting was noted in 4-year-old cherry trees cv. Crystal Champaign grafted on rootstock cv. Adara (Prunus cerasifera L.). Gumming and wilting affected approx. 1% of the production area, leading to the eventual collapse and death of most affected trees within 2-3 years. Discoloration of the vascular system of the crown and roots was also noted. Symptomatic crown and root pieces (0.5 cm) were subjected to surface sterilization: immersed in 1% NaClO for 2 min, rinsed in sterile distilled water, and air-dried in a laminar flow cabinet. Then, plant tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated in a lab bench at room temperature for a week. Cottony and pink colonies were observed growing from the tissues. Two single strains (F175 and F176) were obtained from each tree by excising single spores (Gordon and Okamoto 1991). Isolates produced sparse aerial mycelia with white to pinkish-orange pigmentation on Spezieller Nährstoffarmer Agar (SNA). Both isolates produced microconidia in false heads on short monophialides. Microconidia were hyaline and measured in the range of 5.0-17.5 × 2.5-3.8 µm for both isolates (n = 50). Macroconidia were less abundant, falciform, and hyaline. Morphological characteristics were consistent with identification as Fusarium spp. (Leslie and Summerell 2006). A portion of the translation elongation factor-1 alpha (EF-1α) gene was sequenced using EF1/2 primers (O'Donnell et al. 1998) (GenBank Accession Nos. OR733348 and OR733349). Based on a comparison of 619 base pairs (bp), both isolates exhibited different sequences, with a 99.5% similarity (616/619 bp). A comparison with previously described isolates revealed a 100% match with published F. oxysporum sequences in the GenBank database (KT323846 and MZ404079, respectively). Isolates were used to conduct pathogenicity tests on 1-year-old plants cv. Adara growing in 512 cm3 pots. Using a scalpel, a 6-7 mm-length wound (2-3 mm deep) was made 5 cm above the soil line in all plants. For each isolate, 5 plants were inoculated by placing a 5 mm plug containing 10-day-old mycelia grown in AMAP medium (Borrero et al. 2009) at the incision point. Non-colonized AMAP plugs were used to inoculate 5 control plants. The inoculated sites were sealed with parafilm. Plants were randomly placed in a growth chamber with a temperature of 28/24ºC and a 12-hour photoperiod. A reddish-brown vascular stem discoloration was noticed in all the inoculated plants 73 days after inoculation. On average, the length of the necrotic area was 12.73 cm for F175, 20.12 cm for F176, and 4.59 cm for the control plants. Fusarium oxysporum was successfully re-isolated from all the inoculated plants. Recovered isolates were confirmed to be the same as the inoculated ones by sequencing the EF-1α gene. A one-way ANOVA indicates that plants cv. Adara were susceptible to both F. oxysporum isolates (P < 0.05). This is particularly noteworthy as cherries are predominantly planted on rootstocks and cv. Adara is a widely used rootstock in Spain. While F. oxysporum has been reported as the cause of root and crown rot in sweet cherry (P. avium L.) in British Columbia (Úrbez-Torres et al. 2016), this is the first report of F. oxysporum causing root and crown rot in cherry rootstocks (P. cerasifera L.) in Spain.

First report of Sphaerulina azaleae causing leaf spot on Rhododendron pulchrum Sweet in China.

Rhododendron pulchrum Sweet is a popular ornamental evergreen shrub, renowned for its exquisite flowers. In November 2020, leaf spot disease was observed at Qujing Normal University (25.527°N; 103.744°E), Qujing, Yunnan, China. Symptoms were observed on 25-35 % leaves. This led to early leaf drop, significantly reducing its ornamental value. The infected leaves exhibited irregular, dark brown to blackish spots, with random distribution. To investigate the causal agent, ten symptomatic leaves were collected for pathogen isolation. Leaf spots bearing ascomata were picked with a sterile needle, placed on water agar, and then incubated at 26 ℃ for 24 h. Individual germinated spores were transferred onto PDA for further purification and morphological study. Two pure isolates (chl01 and chl02) were obtained. The colonies on PDA were circular, raised, pink-white at the center, dark brown at the margin, and dark brown to black from reverse after four weeks at 26 °C. Moreover, colonies on PDA sporulated following a 5-hour UV light exposure in a laminar flow hood and a subsequent 14 h light: 10 h dark (14:10) cycle for 5 days at 26 °C. The conidiophores are reduced to conidiogenous cells which are 1.5-3 × 1.5-2.5 µm (n = 10), integrated, hyaline, proliferating sympodially near the apex, cylindrical, and widest at the base. Conidia are hyaline, slightly verruculose, guttulate, cylindrical, straight or irregularly curved, subobtuse at the apex, truncate at the base, and 19-28 × 3-4 µm (n= 30), with 1-3 septa. The morphological features were similar to the genus Sphaerulina (Quaedvlieg et al. 2013). To confirm the candidate pathogen, we performed PCR and gene sequencing using specific primers ACT-512F/ACT2Rd, EF1-728F/EF2, ITS4/ITS5, LSU1Fd/LR5 and fRPB2-5F/fRPB2-414R for actin (ACT), the partial translation elongation factor 1-alpha (TEF1), internal transcribed spacer region (ITS), large subunit ribosomal RNA gene (LSU), and RNA polymerase II subunit (RPB2) genes, respectively (Choi et al. 2020). Sequences were deposited in GenBank with accession numbers OR359635 (ACT), OR359637 (TEF1), OR335339 (ITS), OR335299 (LSU) and OR359636 (RPB2). BLAST searches showed 97.34% (ACT), 99.47% (TEF1), 100% (ITS), 100% (LSU) and 100% (RPB2) identity with those of Sphaerulina azaleae CBS 128605 from GenBank, respectively. A multilocus phylogenetic tree was generated using maximum likelihood (ML) based on the concatenated ACT-TEF-ITS-LSU-RPB2 sequence, placing our isolates within the S. azaleae group with high confidence. To fulfill Koch's postulates, three R. pulchrum plants in the landscape (three leaves per plant) were inoculated by pipetting 0.2 ml spore suspension (1 × 106 conidia/ml) onto the surface of each leaf (Khoo et al. 2022). Three additional plants in the landscape were inoculated with sterile water on similar aged leaves. The leaves were enclosed in plastic bags for 72 hours. At 7 days postinoculation, infected leaves exhibited the symptoms as described above, whereas the controls showed no symptoms. This experiment was repeated twice. The same fungus was reisolated from the infected leaves and identified based on morphology and Sanger sequencing. It is worth noting that S. azaleae can cause leaf spot on Rhododendron yedoense f. poukhanense (Choi et al. 2020). To our knowledge, this is the first report of S. azaleae causing leaf spot on R. pulchrum Sweet in China, indicating that appropriate management strategies are needed.

Exploring the relationships between psychological variables and loot box engagement, part 2: exploratory analyses of complex relationships.

In a pre-registered survey linked to this paper (Exploring the relationships between psychological variables and loot box engagement, part 1: pre-registered hypotheses), we confirmed bivariate associations between engagement with loot boxes (purchasable randomized rewards in video games) and measures of problem gambling, problem video gaming, impulsivity, gambling cognitions, experiences of game-related 'flow', psychological distress and reduced wellbeing. However, these variables have complex relationships, so to gain further insights, we analysed the dataset (1495 gamers who purchase loot boxes and 1223 purchasers of non-randomized content) in a series of Bayesian mixed-effects multiple regressions with a zero-inflation component. The results challenge some well-established results in the literature, including associations between loot box engagement and problematic gambling measures, instead suggesting that this relationship might be underpinned by shared variance with problem video gaming and gambling-related cognitions. An entirely novel discovery revealed a complex interaction between experiences of flow and loot box engagement. Distress and wellbeing are both (somewhat contradictorily) predictive of participants engaging with loot boxes, but neither correlate with increasing loot box risky engagement/spend (among those who engage). Our findings unravel some of the nuances underpinning loot box engagement, yet remain consistent with narratives that policy action on loot boxes will have benefits for harm minimization.

Horizontal laminar flow cabinet with a low background and clean air

The publication presents the design, construction of a prototype, and the course of verification tests of a special horizontal laminar flow cabinet made entirely of plastics. In the cabinet design process, numerical simulations and airflow analyses were used to achieve a laminar, uniform flow in the device's workspace. A prototype was built and subjected to verification tests regarding the intensity and nature of airflow as well as air cleanliness. The cabinet is equipped with its own filtration-ventilation module providing clean air to the workspace and removing used air to the external ventilation system. It ensures an increased level of protection for workers dealing with microorganisms and hazardous airborne chemicals, as well as complete corrosion resistance inside the workspace. A particular area of application for the cabinet is research involving radionuclides, volatile, toxic chemical compounds for which air-recirculating devices cannot be used in the room where they are placed. The developed solution has been protected by industrial property rights and used to implement a contract for the supply of a set of equipment to the laboratory of the Institute of Nuclear Physics of the Polish Academy of Sciences.

Investigating best practice for specimen preparation for biological testing of root canal sealers

IntroductionBiological characterization of root canal sealers is important as it assesses the ability of the root canal sealer to exert antimicrobial properties thus avoiding treatment failures caused by microbial challenge and also assess the cytotoxic effect on the periapical tissues. Assessment of the biological testing of root canal sealers necessitates the sterilisation of the materials prior to evaluation. This study aims to analyse the influence of various sterilisation techniques conducted prior to biological testing on the microstructure and surface properties of endodontic sealers. Assessment of the initial microbial contamination on the material was also undertaken. MethodsFour commercial sealers were investigated. The sealers were either prepared in a laminar flow cabinet or on a laboratory bench top under ambient conditions. Each group was further divided into 5 groups (n = 3) based on the sterilization technique:1) ethanol-10 mins, 2) ultraviolet-1 h, 3) ethanol-10 mins + ultraviolet-1 h, 4) autoclave, and 5) no sterilisation (control). Microbial levels in the materials were assessed by plate streaking technique. The materials were characterized by scanning electron microscopy and energy dispersive spectroscopy, and Fourier transform infrared spectroscopy, before and after sterilisation, to assess any changes in microstructure and chemical composition. ResultsAll the materials did not exhibit contamination when prepared in laminar flow chamber in sterile conditions compared with sealers prepared on the bench top. Three of the commercial materials showed changes in microstructure while one (TotalFill) was not affected by the sterilisation. AH Plus and BioRoot RCS exhibited alterations in water and alcohol peaks in FT-IR while the single syringe sealers (TotalFill and BioRoot Flow) showed no changes. ConclusionsSterilisation methods cause physical and chemical alterations to sealers. Material preparation should be performed in a laminar flow cabinet and a test for sterility should be performed prior to any biological testing being undertaken. If the materials are not sterile, assessment of the effects of the sterilization methods is recommended.

A Novel, Closed-Line Mini Extracorporeal Photopheresis (ECP) Technique Using the Therakos TM CellEx TM Photopheresis System, Developed for Patients with Contraindications Against Conventional ECP Therapy

Introduction: Conventional ECP has proven efficacy for treating several diseases, most significantly steroid-refractory chronic GVHD. Unfortunately, body weight and sufficient venous access limit the availability of this treatment modality. Children, in particular, usually have limited access to ECP. A simplified mini buffy coat ECP technique that allows treating small children and patients with apheresis contraindications has been tested in selected cases during the last 20 years. However, this method relies on an open-line system that requires a longer time and a cell processing laboratory, making this approach challenging. A new -closed-line- method, using the Therakos TM CellEx TM Photopheresis System, has been developed and evaluated in our Center. This method requires no processing laboratory or other special equipment and takes only 60-120 minutes, making the procedure potentially usable in routine practice. Design and methods: A 7-year-old girl had been treated at our Pediatric Bone Marrow Transplantation Unit with grade IV chronic steroid-refractory GVHD (skin, and less severe liver involvement). As all other options were exhausted, ECP became a desirable form of treatment. However, the patient's low body weight and serious venous access problems hindered conventional ECP treatment. We developed a solution that requires 200-250 ml whole blood collection from the patient, takes less than 2 hours, does not need special cell processing equipment, and allows the ECP treatment to commence in the form of a regular autotransfusion. Consent forms were obtained from the patient's caregivers as well as from the local Ethical Committe. The patient's age at the start of treatment was seven years, and her body weight was 28 kg. A total of 17 mini-ECPs were performed weekly up until the submission of this abstract, and the number of treatments per patient was 33. The duration of mini-ECP treatment has been five months. 5 to 8 mL/kg of whole blood was drawn from the patient. Separation of the white blood cell (WBC) rich buffy coat of the patient's blood - with donor blood product supplementation as needed - was performed through an individualized use of the CellEx TM photopheresis kit and blood prime program. The buffy coat was ultraviolet A (UVA)-irradiated after adding 8-methoxypsoralen (8-MOP) within the Therakos TM CellEx TM Photopheresis System and divided into two fractions under a laminar air flow cabinet. The autologous residual blood with the first fraction of the UVA irradiated buffy coat was transfused immediately. The second buffy coat fraction was stored at 4 °C and transfused the following morning through a peripheral vein. Results : The median volume of the collected whole blood was 250 mL (203-250 mL). The median volume was 72 ml (48-88 mL) for the first fraction of the irradiated buffy coat and 71 ml for the second fraction (48- 88 mL). Apoptosis and cell death were analyzed by annexin V staining; apoptosis and lymphocyte proliferation was measured by flow cytometry. UVA irradiation of the 8-MOP infused buffy coat resulted in significant induction of WBC apoptosis at 24 hours. No clinical side effects were observed during mini ECP treatment; all microbiological control tests were negative. Partial response was observed within three months (skin), and the tapering of systemic immunosuppression (methylprednisolone and ruxolitinib) began at five months. Conclusion: Mini whole blood ECP induces apoptosis and lymphocyte proliferation inhibition, both occurring after standard ECP. The closed-line method requires no special equipment and is not demanding for the patient, providing a viable solution to the technical difficulties children with the need for ECP treatment face.

Impact of the implementation of vasoactive drug protocols on costs in the treatment of critically ill patients

ObjectiveTo evaluate the efficiency of the protocolization and centralization of the preparation of intravenous vasoactive drug mixtures in the treatment of critically ill patients. MethodA prospective interventional study (July 2012-December 2014) was conducted to measure the impact of different vasoactive drug protocols on costs in the treatment of critically ill patients. The economic impact was measured by comparing the direct costs (fixed and variable) of the preparation of intravenous vasoactive drug mixtures in the Pharmacy Department with their traditional preparation in hospital care units. The variables time and cost of preparation of an intravenous mixture were measured. Costs included pharmaceutical product, diluent, medical supplies, cost of manpower, and use of laminar flow cabinets in the Pharmacy Department. Costs were measured in Euros. ResultsA statistically significant difference was found between processing times in the Pharmacy Department and those in the hospital care unit (2.10 vs 2.86 minutes). Centralized preparation in the Pharmacy Department was more efficient. The average cost of preparation was €5.24±1.45 in the Pharmacy Department and €5.62±1.55 in the hospital care unit, although this difference did not reach statistical significance. If the analysis had included the cost of intravenous mixtures that had expired prior to their use, the centralized preparation of the mixtures in the Pharmacy Department would have entailed a higher cost (€2 174/y). ConclusionsThe centralized preparation of intravenous mixtures in the Pharmacy Department entails significant time savings compared with their preparation in the hospital care unit.

Flow box decomposition for gradients of univariate polynomials, billiards on the Riemann sphere, tree-like configurations of vanishing cycles for $A_n$ curve singularities and geometric cluster monodromy

The base space of the universal unfolding of A_n -curve singularities is equipped with a stratification such that the geometric monodromy group is generated by wall-crossing mapping classes.

Research on the development of composition and technology of an emulgel with a probiotic component for the treatment of infectious and inflammatory diseases

The aim was conducted research to develop the composition and technology of a new soft preparation under the conventional name "Probioskin" for topical use with a probiotic component in the form of an emulgel.&#x0D; Materials and methods of work. The homogeneity was determined by visual inspection of the experimental samples, using a biological microscope XSP-128 ULAB. The rheological (structural and mechanical) properties of the samples were studied using a Rheolab QC rheoviscosimeter (Anton Paar, Austria) with a C-CC27/SS coaxial cylinder system. The Rheolab QC rheometer is equipped with RheoPlus software. Microbiological studies and biotesting on a biological model of infusoria were performed under aseptic conditions of a laminar flow box (biological safety cabinet AC2-4E1 "Esco", Indonesia) of the Department of Biotechnology of the National University of Pharmacy. The identification of lactobacilli and the number of live cells as critical indicators of a preparation containing live microorganisms were performed at each stage of the study. Bacterioscopic control was carried out by examining smears prepared from samples of the dissolved drug and stained by Gram stain. Bacteriological control was performed by inoculation on thick MRS-4 medium and liquid MRS-1 medium. The number of live lactobacilli (when lactobacilli were co-cultured with the drug components at the stages of development and in the finished product) was determined by surface sowing on Petri dishes with thick MRS-4 medium; after 48 h of incubation at (37±1)°C, colonies grown on the surface of the medium were counted and the number of live lactobacilli expressed in CFU was calculated in 1 ml of the drug.&#x0D; Results and discussion. Based on the conducted research, the composition of a soft preparation for topical use containing dexpanthenol, lactic acid, lyophilized lactobacillus biomass, propylene glycol, peach oil, polysorbate-80, aristoflex AVC, tocopherol, and purified water was developed. The production process of the developed Probioskin product is carried out according to a standardized scheme and consists 8 stages: preparation of raw materials, preparation of oil concentrate of the probiotic component, preparation of aqueous concentrate of active substances, preparation of gel base, preparation of emulsion, dosing into tubes, packaging of tubes into packs, packaging of packs into group containers.&#x0D; Conclusions. On the basis of a complex research, the composition and rational technology of a soft preparation for topical use under the conventional name "Probioskin" in the form of an emulsion was developed.&#x0D; Key words: pharmacological research, soft drug, emulgel, probiotics, dermatological diseases

Feasibility of waste foundry sand in high-strength self-compacting concrete and the effects of elevated temperatures

The disposal and recycling of waste foundry sand (WFS) as industrial solid waste can effectively solve potential environmental pollution problems. This paper conducted an experimental investigation on the effects of elevated temperatures on mechanical properties and microstructural characteristics of high-strength self-compacting concrete (HSSCC) incorporating WFS for substituting river sand with different contents (i.e., 0%, 25%, 50%, 75% and 100% by weight). The workability of fresh concrete including slump flow, J-ring, T500, and V-tunnel box tests were first measured. A series of tests including ultrasound pulse velocity (UPV), residual compressive strength and the behavior of capillary water absorption were conducted before and after different temperatures (100 °C, 200 °C, 300 °C, 400 °C) following an air-cooling time. Further, the thermal analysis and microstructural observation were conducted for the evaluation of high-temperature damage evolution by using thermogravimetry (TG), differential scanning calorimetry (DSC), and scanning electron microscope (SEM). The test results showed that the specimens obtained excellent self-compaction performance at 50% WFS replacement rate. The coupling effect of WFS substitution rate and temperature level has a large effect on the mechanical properties and thermal resistance. Compared with the control group, the 28-day compressive strength of HSSCC-WFS50 was increased by 8.2%. In addition, thermal analysis tests (TG/DTG/DSC) and SEM also elucidated the changes of HSSCC-WFS products and microstructures at high temperature from a fine microscopic perspective to reveal the deterioration mechanism of HSSCC-WFS durability after high temperature damage.