Direct Microscopic Observation Research Articles

A Fluorogenic Pseudoinfection Assay to Probe Transfer and Distribution of Influenza Viral Contents to Target Vesicles.

Fusion of enveloped viruses with endosomal membranes and subsequent release of the viral genome into the cytoplasm are crucial to the viral infection cycle. It is often modeled by performing fusion between virus particles and target lipid vesicles. We utilized fluorescence microscopy to characterize the kinetic aspects of the transfer of influenza viral ribonucleoprotein (vRNP) complexes to target vesicles and their spatial distribution within the fused volumes to gain deeper insight into the mechanistic aspects of endosomal escape. The fluorogenic RNA-binding dye QuantiFluor (Promega) was found to be well-suited for direct and sensitive microscopic observation of vRNPs which facilitated background-free detection and kinetic analysis of fusion events on a single particle level. To determine the extent to which the viral contents are transferred to the target vesicles through the fusion pore, we carried out virus-vesicle fusion in a side-by-side fashion. Measurement of the Euclidean distances between the centroids of superlocalized membrane and content dye signals within the fused volumes allowed determination of any symmetry (or the lack thereof) between them as expected in the event of transfer (or the lack thereof) of vRNPs, respectively. We found that, in the case of fusion between viruses and 100 nm target vesicles, ∼39% of the events led to transfer of viral contents to the target vesicles. This methodology provides a rapid, generic, and cell-free way to assess the inhibitory effects of antiviral drugs and therapeutics on the endosomal escape behavior of enveloped viruses.

Evolution of self-assembled amphiphilic colloidal particles in strong-flavor Chinese baijiu

This study proposed the evolution of self-assembled amphiphilic colloidal particles in Strong-Flavor (SF) Baijiu based on Ostwald ripening for the first time. The evolution process occurs in two stages: disordered amphiphilic molecules self-assemble into small colloidal particles and subsequently undergo Oswald ripening to form larger hydrophobic particles. Microscopic observations revealed the average size of oil-like spherical colloidal particles in Baijiu increased from 1.86 μm to 2.96 μm while the number of particles decreased by 39.50% during the 16-year cellaring process of SF Baijiu, consistent with the particle size trend observed via laser scattering. During fusion process, the charge-to-mass ratio of positively charged colloidal particles decreased, leading ζ-potential decreased from 23.7 mV to 4.66 mV within 16 years of storage. The electrochemical impedance spectroscopy approach tracked the unidirectional variation in the dielectric constant during evolution of SF Baijiu, reflecting the gradual expansion of colloidal particles, which aligns with the evolution trend observed in molecular dynamics simulations. By integrating direct microscopic observations of amphiphilic colloidal particles with electrochemical techniques, the evolution of Baijiu samples is capable to be evaluated in-situ, laying the foundation for intelligent Baijiu aging monitoring technology.

Anodic oxidation of wireless niobium electrode in bipolar electrochemistry

The anodic oxidation of a wireless niobium substrate serving as a bipolar electrode (BPE) in a direct or alternating current electric field was investigated in detail via spectrophotometry, electrochemical measurement, glow discharge–optical emission spectroscopy, and direct microscopic observation. Niobium oxide was a suitable material for the experiment because it is amorphous and can be evaluated through conventional electrochemical methods. It also has a clear interference color, enabling easy optical evaluation. The effect of the BPE configuration (e.g., vertical and horizontal alignments) in the cell on the anodic oxidation area was also investigated visually using the interference color. Transmission electron microscopy observation was also performed for an accurate measurement of the thickness of the anodic film formed on the niobium BPE. Although the basic film formation behavior and film composition were consistent with those in conventional anodization, even in wireless operation, the effective voltage directly contributing to film formation was found to be lower than the applied voltage (30%–70% of the applied voltage), depending on the electrolysis conditions.

Evaluation and validation of a PrintrLab-based LAMP assay to identify Trypanosoma cruzi in newborns in Bolivia: a proof-of-concept study

Vertical transmission of Trypanosoma cruzi represents approximately 20% of new Chagas disease cases. Early detection and treatment for women of childbearing age and newborns is a public health priority, but the lack of a simple and reliable diagnostic test remains a major barrier. We aimed to evaluate the performance of a point-of-care loop-mediated isothermal amplification (LAMP) assay for the detection of Tcruzi. In this proof-of-concept study, we coupled a low-cost 3D printer repurposed for sample preparation and amplification (PrintrLab) to the Eiken Tcruzi-LAMP prototype to detect vertically transmitted Tcruzi, which we compared with standardised PCR and with the gold-standard algorithm (microscopy at birth and 2months and serological study several months later). We screened pregnant women from two hospitals in the Bolivian Gran Chaco province, and those who were seropositive for Tcruzi were offered the opportunity for their newborns to be enrolled in the study. Newborns were tested by microscopy, LAMP, and PCR at birth and 2months, and by serology at 8months. Between April 23and Nov 17, 2018, 986mothers were screened, among whom 276were seropositive for Tcruzi (28·0% prevalence, 95% CI 25·6-31·2). In total, 224infants born to 221seropositive mothers completed 8months of follow-up. Congenital transmission was detected in nine of the 224newborns (4·0% prevalence, 1·9-7·5) by direct microscopy observation, and 14more cases were diagnosed serologically (6·3%, 3·6-10·3), accounting for an overall vertical transmission rate of 10·3% (6·6-15·0; 23of 224). All microscopy-positive newborns were positive by PrintrLab-LAMP and by PCR, while these techniques respectively detected four and five extra positive cases among the remaining 215 microscopy-negative newborns. The PrintrLab-LAMP yielded a higher sensitivity than microscopy-based analysis. Considering the simpler use and expected lower cost of LAMP compared with PCR, our findings encourage its evaluation in a larger study over a wider geographical area. Inter-American Development Bank.

The Relative Frequency of Histomonas meleagridis Infection in Turkey Flocks in Some Provinces of Iran

Background: Histomoniasis is caused by the protozoan Histomonas meleagridis with an intermediate host of Heterakis gallinarum, which results in ulceration of the ceca walls, enlargement of the ceca by large casts, mesenteric inflammation, and liver necrosis. This disease is very important in Iran’s growing turkey breeding industry. Objectives: The present study aims to evaluate the relative frequency of H. meleagridis infection in different turkey flocks to draw a cross-sectional picture of H. meleagridis infection in Golestan, Mazandaran, Guilan, and Tehran provinces of Iran. Methods: This study is a cross-sectional survey of H. meleagridis infection during spring. Dropping samples were taken from backyard and commercial turkey flocks. After taking the fecal samples, they were investigated by Giemsa staining under a light microscope. A PCR test was performed to confirm the diagnosis of infection. Results: Out of 240 samples (from 19 flocks), 20 infected samples were detected by direct microscopic observation of H. meleagridis, and PCR confirmed 15 samples. Conclusion: The results of this study showed that the relative frequency of H. meleagridis infection was lower than in similar studies in other parts of the world. This finding may be due to Iran’s less widespread use of turkey production. Considering the growth of the turkey production industry in Iran over the last decade and its further production over the next few years, it is necessary to evaluate histomoniasis.

Risk Factors for Candidiasis among Patients Referred to Medical Mycology Laboratory of the Special Clinical of Kermanshah University of Medical Sciences

Introduction: Candidiasis is undoubtedly one of the most important and common diseases of opportunistic fungi in human. Factors such as: age, physiological changes, long-term use of antibiotics, diabetes, immune deficiency, cardiovascular disease can make a person prone to candidiasis. In this study, risk factors contributing to candidiasis in the patients referred to the mycology laboratory of the special clinic of Kermanshah University of Medical Sciences in 2013 to 2018 were investigated. Methods: In a descriptive study, to show the effective factors in the development of candidiasis as an opportunistic fungal disease, all patients during 2013 to 2018 that referred to the mycology laboratory of the special clinic of Kermanshah University of Medical Sciences were examined. In this study, demographic characteristics such as age, gender, occupation, and residence were collected. The results of direct microscopic observation and mycological culture of patient samples were determined. The results were expressed as descriptive statistics. Results: In this study, 26.98% of 541 people had cutaneous candidiasis, 72.08% had nail candidiasis, and 0.92% had systemic candidiasis. The most common species is Candida albicans. The highest frequency of skin candidiasis is related to the age group of 40-49 years (20.54%). It was more common in patients with a history of cardiovascular disease and diabetes and 47% of patients mentioned excessive contact with water. The highest frequency of nail candidiasis is related to the age group of 30- 39 years and women. It is more common among housewives. It is more prevalent in patients with a history of cardiovascular disease and diabetes, respectively. 75% of patients with systemic mucosal candidiasis had immunodeficiency. Conclusion: The most important factor in preventing candidiasis is public health. For age group of 20-29 years, when suffering from chronic diseases such as diabetes, cardiovascular and immunodeficiency disease.

Identification of Ceramic Traditions on the Prehistoric Mines of Gavà (Barcelona, Spain)

Abstract In recent years, studies focused on Chaîne Opératoire reconstruction of pottery vessels have shown important developments and provided new data related to vessel production and the complexities acquired by prehistoric societies. This research focuses on the pottery vessels discovered at the Mines Prehistóriques de Gavà (Barcelona, Spain), which constitute the earliest evidence of a mining center for variscite during the 4th millennium BC. The main objective of this study is to determine the technological procedures employed in the production of these potteries by identifying the Chaînes Opératoires and establishing their relationship to the site’s context, which is linked to extensive exchanges within the European network. Through a meticulous analysis and comparison of the morpho-typological and technological characteristics of the vessels, we aim to identify different production methods. The findings of this study, along with their reconstruction in techno-stylistic trees, contribute to a deeper understanding and explanation of the variability and technological complexity found in these pottery vessels. This work exposes a novel approach to studying the pottery vessels in Iberian Peninsula by involving the application of archaeometric techniques such as direct macroscopic and microscopic observation traces (using naked eyes; microscopic Dino – Lite), as well as chemical analyses such as petrographically and X-ray diffraction enabling the identification of key mineral components within the clay.

Experimental study on the influence mechanism of clay particles on the microbial treatment of granite residual soil

The content of clay particles has a key influence on the physical and mechanical properties of microbial solidified granite residual soil. Microbial-induced calcite precipitation (MICP) treatment method of “mixing + grouting” was used to prepare microbial solidified granite residual soil samples with different clay contents and to explore the influence of clay content on the reinforcement effect of microbial solidified granite residual soil. A series of direct shear tests, disintegration tests, and microscopic observation tests was conducted to quantitatively analyze the influence of clay content on the shear strength and disintegration resistance of microbial solidified granite residual soil. The conclusions are summarized as follows: the shear strength and disintegration resistance of granite residual soils solidified by MICP treatment were remarkably improved, with the shear strength increasing by 9%∼16% when the clay content varied from 10%∼40%. The internal friction angle of the granite residual soil with different clay contents increased and then decreased with the increase in clay content. The cohesion of the microbial solidified granite residual soil increased with the increase in clay content, and the internal friction angle reached a minimum of 20% clay content (17.27°), which is 31.85% lower than that of 0% clay content (25.34°). The cohesion of the microbial solidified granite residual soil reached a maximum of 40% clay content (39.54 kPa), which is 8.69 times higher than that of 0% clay content (4.55 kPa). The MICP treatment technology is capable of effectively improving the shear strength and disintegration resistance of granite residual soils. The MICP treated process produces calcium carbonate precipitates that link the sand particles and fill the pores between the soil particles, increasing the number of larger agglomerates in the granite residual soil, which is the essential reason for the improvement in the softening and disintegration of the granite residual soil when exposed to water.

Base‐assisted anionic polymerization of alkyl sorbates and subsequent ring‐closure without extreme dilution for synthesis of cyclic stereoregular poly(alkyl sorbates)

AbstractA typical base, lithium diisopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS), was found to initiate the anionic chain‐growth polymerization of ethyl sorbate (ES) in the presence of an aluminum Lewis acid, that is, methylaluminum bis(2,6‐di‐tert‐butyl‐4‐methylphenoxide) (MAD) and, after ES consumption, ring‐closure occurred without highly dilute conditions to give the cyclic poly(ES). The finding of ring closure was supported by matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectroscopy and direct transmittance electron microscope (TEM) observations, in which a series of spectral patterns separated by the mass of ES and cyclic objects with 7.4–25.9 nm rings were observed, respectively. The results indicated that instead of the use of an expensive N‐heterocyclic carbene (NHC), employing these commodity bases to initiate chain polymerization enabled us to fabricate the intended cyclic vinyl polymers without highly dilute conditions. Also, this polymerization proceeded even at −80°C to give threo (98%)‐disyndiotactic (91%) stereoregular poly(ES) in an excellent yield with narrow molecular weight distributions (Mn = 5.9 × 103–8.4 × 103, Mw/Mn = 1.14).

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 µL drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 °C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Degradation of polyacrylamide (PAM) in aqueous solution by electron beam technology: Efficiency, viscosity reduction and correlation with microstructure of polymer molecules

In this paper, we present a systematic evaluation about degradation of partially hydrolyzed polyacrylamide (HPAM) in aqueous solution by ionizing radiation to find relationship between HPAM decomposition, viscosity reduction and microstructure of polymer molecules. The viscosity of the two HPAM solution declined rapidly from 5.2 to 3.0 mPa s to 1.4–1.2 mPa s with the adsorbed dose of 0.1 kGy, and then reached the level of pure water at 0.5 kGy. The molecular weight declined remarkably from 1.6 × 107 Da to 1.4 × 105 Da (HPAM1) and from 1.2 × 107 Da to 1.1 × 106 Da (HPAM2) at 0.1 kGy. The HPAM solution changed from pseudoplastic fluid behavior to Newtonian fluid after irradiation. Following ionizing radiation, the presence of anion Na+, Ca2+, Mg2+, anions HCO3- and SO42-(1000–5000 mg/L) and surfactant sodium dodecyl sulfate (SDS) (50–100 mg/L) respectively has slight effect on the rate and extent of viscosity reduction, while CO32- and surfactant petroleum sulfonate exhibit a great negative effect on reducing HPAM viscosity. Ionizing radiation is able to destroy the molecular structure and spatial structure of HPAM. Both the C-C backbones and pendant are attacked. It is proposed that the degradation products include ammonium, aliphatic hydrocarbon, small molecular acids and compounds with double-bond. With the absorbed dose of 0.1 kGy, the micelles-like aggregates are broken into small particles by direct microscope observation, while the stretched and coiled molecular strands shrink and curl to form an irregular network from AFM and SEM observation. The chains break into fragment scattered randomly as increasing dose to 1.0 kGy. Both the diameter size and dispersity of HPAM molecules decreased remarkably after irradiation.·OH is the major active species responsible for HPAM degradation and viscosity reduction. This study contributes to a multiscale understanding the mechanism of ionizing radiation-induced degradation of HPAM in aqueous solution.

Experimental phase diagram for the PVDF – DMAc– water ternary system with new topology: Method of construction, thermodynamics, and structure formation of membranes

Ternary mixtures of a semicrystalline (SC) polymer with a solvent and a nonsolvent are widely used primarily for preparation of membranes by nonsolvent induced phase separation (NIPS). A detailed analysis of the published studies devoted to phase equilibrium in such systems showed that the phase diagrams were either incomplete or presented in a schematic form. In the present paper, we propose a new method of plotting the ternary phase diagrams for the SC polymer – solvent – nonsolvent mixtures. This method is based on finding the location of the isotherm intersection with the curves on the temperature–composition pseudobinary phase diagrams for the SC polymer mixtures with solvent and binary mixtures of solvent and nonsolvent of different composition. The coordinates of the intersection points were used to plot for the first time the complete ternary phase diagram for the widely used mixture of polyvinylidene fluoride (PVDF) with dimethyl acetamide (DMAc) and water. The curves on the phase diagram separate the regions of homogeneous mixtures, coexistence of two liquids, macroscopically uniform gels (swollen SC polymer) and coexistence of such gels with liquid. Using the plotted phase diagram, a new understanding of the NIPS mechanism was proposed and confirmed by direct microscopic observation of the NIPS process and SEM analysis of the membranes obtained following this way. An unexpected result of the study is discovery of the crystalline complexes in the mixtures of PVDF and DMAc, which was confirmed using both DSC and XRD techniques.

#3482 IN VIVO EVALUATIONS OF THE PROTECTIVE EFFECTS OF NEW FORMULATIONS FOR PERITONEAL DIALYSIS ADOPTING A POWERFUL MULTI-PHOTON MICROSCOPY BASED APPROACH

Abstract Background and Aims Peritoneal dialysis (PD) is a renal replacement therapy that enables metabolic waste products and excess fluids to be eliminated through the peritoneal membrane. Exposure to conventional dialysates at high glucose concentrations is considered critical for the pathogenesis of peritoneal fibrosis, angiogenesis, and epithelial-mesenchymal transition (EMT). These events largely contribute to peritoneal membrane aging, resulting in ultrafiltration and clearance failure. Pre-clinical research in this field suffers from the lack of valid and reproducible in vivo systems and so far has been limited to in vitro systems of mesothelial or endothelial cell lines. In vitro studies on mesothelial cell monolayers have validated the significant protective effects of dialysates with alternative osmo-metabolic agents. With their better biocompatibility these novel formulations could provide a valid substitute for conventional solutions (1,2). The method proposed, based on multi-photon microscopy, aims to study the physiology of the peritoneal membrane during dialysis exchange and to validate the effects of biocompatible dialysates in animal models of fibrosis as observed in the course of dialysis treatment. Method We have implemented a surgical procedure to optimize the stability of a flap of parietal peritoneum to allow direct microscope observation. The innovative technology of multi-photon microscopy enables one to observe the vessels of microcirculation in vivo and provides a 3D rendering of the peritoneal membrane, giving an overview of all the single layers without the use of specific markers (Figure 1). One may also assess specific phenomena induced by the fibrotic process, such as the thickening of the sub-mesothelial interstitium, exploiting the autofluorescence signal from excited collagen fibers caused by the physical phenomenon of Second Harmonic generation. In vivo microscopy evaluation of peritoneal membrane senescence parameters was conducted in rats subjected to one daily intraperitoneal injection (10 mL/day) for 15 days with one of the following PD solutions: a commercially available glucose-based neutral-pH, low-GDP, PD solution (Physioneal 3.86%, Baxter Healthcare, Italy), and a new single-chamber low glucose PD solution containing osmo-metabolic agents such as L-carnitine and xylitol (XyloCore HS, Galenica Senese, Italy). XyloCore HS is a lactate-buffered PD solution with glucose (1.5%), L-carnitine (0.02%) and xylitol (2.0%). The osmotic strength of the two PD solutions tested was comparable. Results Treatment with XyloCore HS was associated with a significant reduction in thickness of the sub-mesothelial interstitium (p = 0.013), the density of collagen fibers (p = 0.012) and the vascular composition (p = 0.006), as well as the number of branch points (p = 0.0335), when compared to rats treated with a commercial glucose-based PD solution, Physioneal. Figure 2 shows the different thicknesses of sub-mesothelial stroma upon treatment with the PD solutions under investigation. Conclusion Previous in vitro studies have shown that XyloCore (1,2), a novel glucose-sparing PD solution currently in Phase III clinical development (ELIXIR - NCT03994471), is able to counteract the glucotoxic effects on the peritoneal membrane induced by conventional dialysates. Our pre-clinical in vivo methodology not only confirms our previous in vitro findings, but also suggests that long-term protective effects may be achieved with XyloCore. Further studies are in progress in a diabetic animal model to extend the favorable peritoneal in vivo findings of XyloCore treatment to a systemic level, possibly by improving glycemic control.

Identification of a new Sarcocystis sp. in marsh deer (Blastocerus dichotomus) from wetlands of Argentina.

The marsh deer (Blastocerus dichotomus) is the largest South American native deer species and is listed as "Vulnerable" by IUCN due to the population reduction. As part of a conservation and disease surveillance program, muscle samples from 14 marsh deer found dead in 2016 and 2017 in northeast Argentina were obtained at necropsy. Samples from each animal were processed as pooled muscles (heart, diaphragm, tongue and hindlimb) by homogenization and direct microscopical observation to detect intracellular Sarcocystis spp. cysts. Sarcocysts were observed in six samples, and several cysts recovered from two samples were processed by transmission electron microscopy. The cysts were thin-walled and showed a cyst-wall ultrastructure with ribbon-like protrusions similar to other species using cervids as intermediate host and canids as definitive hosts. Genomic DNA from individual sarcocysts from three marsh deer were successfully amplified by PCR of 18S rRNA and COI gene fragments and further sequenced. Sequence comparison revealed a 99.3-100% identity among them and only 93.7-96.6% and 88.8-89.7% identity at 18S rRNA and COI markers, respectively, with other Sarcocystis spp. Despite morphological similarities, the high sequence divergence at 18S rRNA and COI fragments allowed the assumption that Sarcocystis sp. from marsh deer is a different species from others using cervids as intermediate hosts. Therefore, we propose the name Sarcocystis blastoceris n. sp. for the species infecting marsh deer.

Isolation and Identification of Bacterial Communities in Neutral Mine Drainage in Central Slovakian Neovolcanites (Slovakia)

There are several sources of mine drainage left over from past mining sites in the Central Slovakian neovolcanites. The neutral pH and high concentrations of sulphates and multiple potentially hazardous elements, such as zinc or manganese, are typical in this region. However, this environment could be home to specific microbiota. The aim of the study was to characterize bacterial populations in mine drainage in the Central Slovakian neovolcanites. Direct microscopic observations, cultivation methods, MALDI TOF mass spectrometry, and 16S rRNA gene sequencing of isolates were used for identification. Gram-positive and Gram-negative bacteria were almost equally represented in the mine water samples. The most abundant bacterium was the genus Bacillus spp. (43.48%). Another large group of bacteria consisted of Proteobacteria (34.78%), represented by Pseudomonas spp. (17.39%), Serratia spp. (13.04%), and Providencia spp. (4.35%). Our data confirm the presence of Bacillus spp. and Pseudomonas spp. as bacterial species occurring in an environment polluted by potentially hazardous elements, which may indicate their bioremediation potential.

Phylogenetic analysis of pathogenic Candida spp. in domestic pigeons.

The current study was conducted to survey the prevalence of pigeon candidiasis in diseased pigeons suspected to candidiasis by isolation, microscopic examination, and polymerase chain reaction (PCR) method and to characterize Candida spp. phylogenetically. For this purpose, samples were obtained from 100 suspected pigeons from September 2018 to February 2019 in Ahvaz, Iran. Cloacal and oropharyngeal swab samples were collected from each diseased pigeon with diarrhea resistant to the antibiotics, crop stasis, white diphtheritic membrane in the mouth, regurgitation, and vomiting. Sabouraud dextrose agar was used as a culture medium. Selected colonies were stained with lactophenol cotton blue stain. In the culture and direct microscopic observation, 19.00% of birds were suspected to candidiasis. Twenty-two isolates were identified. All 22 isolates were confirmed as Candida spp. By PCR method. The PCR test confirmed the presence of Candida spp. in 19.00% of pigeons. Based on the sequencing results of some PCR products, the isolates belonged to Candida albicans and Candida glabrata. The results revealed a 99.78% accordance when compared with other sequences of C. albicans which were formerly deposited in GenBank® from Colombia, Indonesia, China, and Sudan. The results revealed a 99.54% accordance when compared with other sequences of C. glabrata which were formerly deposited in GenBank® from the Netherlands and Spain. The symptoms such as diarrhea resistant to antibiotics, crop stasis, white diphtheritic membrane in the mouth, regurgitation, and vomiting were the most prevalent clinical symptoms in positive pigeons.

Relationships between the seasonal dynamics of soil fungi biomass and environmental factors in predominating forest types in the Bryansk woodlands (European Russia)

Being the crucial part of the forest soil's microbial pool, soil fungi in general and mycorrhizal fungi in particular are an important study object when it comes to forest ecosystems sustainability and preservation. Thus, the study of ectomycorrhizal fungi has been carried out in the Bryanskiy Les State Nature Biosphere Reserve, located in the south-eastern part of the Bryansk woodlands (European Russia). Forest types featured in the study are the local predominating types, namely green-moss-fructiculose pine forests and polydominant deciduous broadleaved nemoral-herbaceous forests with spruce. This study was aimed to assess seasonal dynamics of soil fungi' biomass overall and ectomycorrhizal fungi in particular over the course of the 2017 vegetation period (May – November) and its dependence on biotic and abiotic environmental factors, such as soil water content, temperature and vegetation. The vegetation period was divided into three periods of observation, namely an early (May – July), middle (July – September) and late (September – November) one. The method used to assess the fungal biomass was direct microscopic observation using the fluorescein diacetate staining. In order to estimate the ectomycorrhizal fungi biomass separately, trenching and in-growth mesh bags were employed. The obtained results suggest that the soil fungi biomass steadily increases over the vegetation period in both studied forest types. This is mostly affected by the forest type, available water amount and seasonal changes, while the temperature's impact is less pronounced. On average, the soil fungi biomass was higher in broadleaved forests than in pine forests (2.288 mg C × g-1 soil vs. 1.672 mg C × g-1 soil, respectively), with non-ectomycorrhizal component having comparable biomass. The dynamics of biomass differed in the two forest types. However, noticeable differences (p < 0.1) between the two forest types have only been recorded during the July – September period. The biomass of ectomycorrhizal fungi is smaller than the biomass of non-mycorrhizal fungi, but at the same time it is less affected by changes in moisture. Besides that, the study has shown that the forest litter characteristics can greatly affect the dynamics of the fungal biomass.

Abstract 15305: The Dynamic Behavior of Polymer Microbubbles During Long Ultrasound Tone-Burst Excitation and Its Application for Sonoreperfusion Therapy

Objective: Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC) mediated therapies have been shown to restore perfusion and enhance drug/gene delivery. Due to the potentially longer circulation time and relative ease of storage and reconstitution of polymer shelled MBs compared with lipid MBs, we investigated the dynamic behavior of polymer MBs and its therapeutic potential for sonoreperfusion (SRP) therapy. Methods: We explored the fate of polymer MBs during a single long tone-burst exposure (1 MHz, 5 ms) at various acoustic pressures (0.25-1.5 MPa) and MB concentrations (2х10 6 -2х10 8 MB/mL) via direct high-speed microscopy observation and passive cavitation detection (PCD) and compared with lipid MBs. SRP efficacy of these MBs was investigated in an in vitro constant flow system at various pulse durations (1 MHz, 1-5 ms every 3 sec for 20 min) and acoustic pressures (0.6-1.5 MPa). The pressure differential across a porous mesh partially occluded with microthrombi was recorded as a function of time during treatment and used as a measure of thrombus burden. The terminal pressure drop and the initial rate of pressure drop were derived and compared. Results: Both polymer and lipid MBs first underwent stable or inertial cavitation depending on the acoustic pressure, and then formed gas-filled clusters that continued to oscillate, fragment, and form new gas-filled clusters during the single US burst. PCD confirmed continued acoustic activity throughout the 5-ms US excitation. SRP efficacy with polymer MBs increased with the pulse duration and acoustic pressure ( p <0.05, ANOVA), similarly to that of lipid MBs, but no significant differences were found between polymer and lipid MBs ( Figure 1 ). Conclusion: Under the acoustic conditions tested, both polymer and lipid MBs demonstrate similar thrombolytic effects in vitro suggesting that persistent cavitation activity from polymer MBs during long tone-burst US confers excellent reperfusion efficacy.

MHC II – EGFP knock-in mouse model is a suitable tool for systems and quantitative immunology

Immunology is a rapidly evolving field of research with sophisticated models and methods. However, detailed data on total immune cell counts and population distributions remain surprisingly scarce. Nevertheless, recently established quantitative approaches could help us understand the overall complexity of the immune system. Here, we studied a major histocompatibility complexclass II – enhanced green fluorescent protein knock-in mouse model to precisely identify and manipulate lymphoid structures. By combining flow cytometry with light sheet microscopy, we quantified MHC II+ populations of the small intestine and associated individual mesenteric lymph nodes, with 36.7 × 106 cells in lamina propria, 3.0 × 105 cells in scattered lymphoid tissue and 1.1 × 106 cells in Peyer's patches. In addition to these whole-organ cell counts, we assessed approximately 1 × 106 total villi in the small intestine and 450 scattered lymphoid tissue follicles. By direct noninvasive microscopic observation of a naturally fully translucent mouse organ, the cornea, we quantified 12 ± 4 and 35 ± 7 cells/mm2 Langerhans- and macrophage-like populations, respectively. Ultimately, our findings show that flow cytometry with quantitative imaging data analysis enables us to avoid methodological discrepancies while gaining new insights into the relevance of organ-specific quantitative approaches for immunology.

Distribution of Diatom Resting Stages in Sediment near Artificial Reefs Deployed in the Dysphotic Zone: Exploration of New Artificial Reef Function

Artificial reefs (ARs) have multiple beneficial effects involved in increased fishery production. The present study investigated diatom resting stages in the sediments near ARs at a depth of about 90 m by the combined use of direct microscopic observation and the MPN method, seeking a novel function of AR deployment that potentially increases biological production. Although the concentration of diatom resting stages at A2 (north end station of the AR area), composed mainly of Chaetoceros, was ten times the concentration at A5 (non-reef station), the concentration at A1 (south end station of the AR area) was the same level as at A5. These results suggest that the AR contributes to the accumulation of diatom resting stages while the degree of accumulation is attributed to the arrangement of ARs and their physical environment such as flow directions. Interestingly, live pennate diatoms possessing photosynthetic pigments were constantly observed in all samples despite the low light intensity (≤1.2 µmol photons m−2 s−1), which provides new ecological insight into their biological and physiological characteristics. In this study, the distribution of viable diatom resting stages and live pennate diatoms in the sediment around ARs in a dysphotic zone was revealed for the first time, casting a light on the novel function of ARs potentially enhancing primary production around them.