Optical Density Research Articles

Clinical and morphological features of the myometrium in patients with placental adherent and invasive pathology

BACKGROUND: The fast increase in the frequency of placental adherent and invasive pathology worldwide accounts for the growing interest in studying the pathogenesis of placenta accreta. According to the literature, the clinical and morphological features of the myometrium from the placental attachment area in placental adherent and invasive pathology are described in single articles. Study of morphological features using proteolytic markers in the myometrium from the placenta accreta area could help in understanding the pathogenesis of placental adherent and invasive pathology. For the first time in this study, the clinical and morphological features of the myometrium from the placental attachment area in patients with uterine scar and placental adherent and invasive pathology are compared with the myometrium of women with uterine scar without placenta accreta and intact myometrium. AIM: The aim of this study was to evaluate the expression of matrix metalloproteinase 2 and tissue inhibitors of matrix metalloproteinases 1 and 2 in myometrial biopsies in placental adherent and invasive pathology. MATERIALS AND METHODS: This study included 15 myometrial biopsies from the placental site, which were divided into three groups according to the clinical diagnosis of the patients: group 1 (main group), with a uterine scar after cesarean section and placental adherent and invasive pathology (n = 5); group 2 (comparison group), with a uterine scar after cesarean section (n = 5); group 3 (control group), with a normal pregnancy without a uterine scar (n = 5). Histological examination was carried out using the standard procedure. Immunohistochemical study was performed using antibodies to matrix metalloproteinase 2 and tissue inhibitors of matrix metalloproteinases 1 and 2 (Abcam, USA). Morphometry was carried out using the VideoTesT-Morphology 5.2 program (Videotest Ltd., Russia). The statistical analysis was performed using the IBM SPSS Statistics 26.0 software. RESULTS: In the biopsies of the main group, we verified terminal chorionic villi with hypervascularization and uneven plethora of the vascular bed among hypertrophied muscle fibers, a basal plate with lumen ectasia of unevenly full-blooded vessels, and the absence of a decidual membrane, in contrast to groups 2 and 3. The matrix metalloproteinase 2 expression area in the main group was higher than in the comparison and control groups (p = 0.008; p1–2 = 0.049*, p1–3 = 0.011), the matrix metalloproteinase 2 optical density not differing between the groups (p = 0.122). The tissue inhibitors of matrix metalloproteinases 1 expression area was higher in the comparison group compared to the main group (p = 0.035; p2–3 = 0.032), and the tissue inhibitors of matrix metalloproteinases 1 and 2 optical density was higher in the comparison group compared to control (p = 0.008; p2–3 = 0.005). CONCLUSIONS: In myometrial biopsies of patients with a uterine scar and placental adherent and invasive pathology, we verified pathology of the basal plate of the placenta, increased matrix metalloproteinase 2 expression and decreased tissue inhibitors of matrix metalloproteinases 1 expression, which may indicate the peculiarities of the inflammatory response in the myometrium in placental adherent and invasive pathology.

Improved Process Stability and Light Detection in Phototransistors via Inverted MoS2/a‐IGZO Heterojunction Integration

The integration of molybdenum disulfide (MoS2) with other semiconductors to form heterojunction phototransistors demonstrates potential for optoelectronic applications due to their strong interaction with light and tunable bandgaps. Conventionally, bottom‐gate phototransistors based on MoS2 and amorphous indium–gallium–zinc oxide (a‐IGZO) are fabricated by deposition of the a‐IGZO film on the gate dielectric, followed by deposition of the MoS2 film, resulting in a MoS2/a‐IGZO heterojunction. In this study, an inverted MoS2/a‐IGZO (or denoted as a‐IGZO/MoS2) heterojunction integration for bottom‐gate phototransistors, where the MoS2 film is deposited on the gate dielectric first, followed by the deposition of the a‐IGZO film, is presented. This configuration is designed to enhance the processability and electrical properties of the phototransistor. Under visible light exposure with a low optical power density of 5.3 μW cm−2, the a‐IGZO/MoS2 heterojunction phototransistor demonstrates significantly improved photoresponsivity (1.6, 2.8, and 17 A W−1 at 635, 520, and 405 nm wavelengths, respectively) compared to the MoS2 single‐channel phototransistor. Time‐dependent photoresponse measurements reveal that the a‐IGZO/MoS2 heterojunction phototransistor responds more quickly to light changes and generates a tenfold‐greater photocurrent than that of the MoS2 single‐channel phototransistor.

Crosslinkable Ligands for High-Density Photo-Patterning of Perovskite Nanocrystals.

Perovskite nanocrystals (PNCs) are promising luminescent materials for electronic color displays due to their high luminescence efficiency, widely-tunable emission wavelengths, and narrow emission linewidth. Their application in emerging display technologiesnecessitates precise micron-scale patterningwhile maintaining good optical performance. Although photolithography is a well-established micro-patterning technique in the industry, conventional processes are incompatible with PNCs as the use of polar solvents can damage the ionic PNCs, causingsevere luminescence quenching. Here,we report the rational design and synthesis of a new bidentate photo-crosslinkable ligand for the direct photo-patterning of PNCs. Each ligand contains two photosensitive acrylate groups and two carboxylate groups, and is introduced to the PNCs via an entropy-driven ligand exchange process. In a close-packed thin film, the acrylate ligands photo-polymerize and crosslink under ultraviolet light, rendering the PNCs insoluble in developing solvents. A high-density crosslinked PNC film with an optical density of 1.1 is attained at 1.4µm thickness, surpassing industry requirements on the absorption coefficient. Micron-scale patterning is further demonstrated using direct laser writing, producing well-defined 20µm features. This study thus offers an effective and versatile approach for micro-patterning PNCs, and may also be broadly applicable to other nanomaterial systems.

Maillard Type Reaction for Electroless Copper Plating onto Ceramic Nanoparticles

A high demand for oxide dispersion strengthened (ODS) materials arose in wide fields of application. The electroless plating method has been presented as an elegant way to overcome different surface energies and obtain metal‐plated ceramics. However, copper electroless plating is still performed under harsh conditions with toxic and expensive reagents, including noble metals and harsh reducing agents like formaldehyde and hydrazine. To create a pure copper metal matrix in an environmentally friendly and efficient way a previously reported method is advanced in a way that naturally occurring amino acids are utilized. A screening of several amino acids leads to an improvement in phase purity and atomic efficiency. Therefore, a Maillard type reaction with lysine as amino compound is reported to show the best results in the particle coating for all ceramic nanoparticles evaluated. The metal plating results in uniform round micro particles showing a homogeneous coating. In order to obtain copper matrix composites, the prepared particles are successfully implemented in a PBF‐LB/M process after mixing with a highly conductive copper alloy. The resulting products were investigated by transmission electron microscopy, scanning electron microscopy, powder X‐ray diffraction, and optical density measurements.

Combined optical measurement of dissolved oxygen tension (DOT), pH value, biomass and viscosity in shake flasks

Shake flasks are widely spread in microbial process development. Characterization of the processes by manual offline sampling is time-consuming, highly laborious and a contamination risk. Online monitoring of key parameters would provide deeper insights, while saving time and effort. In this study, a device for optical online monitoring of dissolved oxygen tension (DOT), biomass, pH value and viscosity in shake flasks is presented. DOT measurement relies on fluorescent oxygen sensitive nanoparticles. The fluorescence intensity signal of the nanoparticles is used to trigger the DOT and scattered light measurements inside the rotating bulk liquid. The scattered light signal (610 – 630 nm) can be correlated to offline measured optical density OD600, even at elevated viscosity. The pH value is monitored online by using pH sensor spots, fixed inside the shake flasks. The shift of the angle of the bulk liquid Θ-Θ0 is correlated to the offline measured viscosity. Detection of the leading edge of the bulk liquid, necessary for viscosity measurement, can be performed either using the fluorescence intensity signal of the oxygen nanoparticles or the scattered light signal.

Enhanced Growth and Productivity of Arthrospira platensis H53 in a Nature-like Alkalophilic Environment and Its Implementation in Sustainable Arthrospira Cultivation

Synthetic culture media, such as Zarrouk’s medium (ZM), are widely used in industrial Arthrospira cultivation but rely heavily on chemical fertilizers, raising concerns over cost and environmental impact. In natural habitats where Arthrospira blooms, the macronutrient concentrations are much lower than those provided by synthetic media. We hypothesized that natural growth may be facilitated by a microbial consortium. To test this, we developed a lab-scale Arthrospira platensis H53 cultivation system using a newly developed organic compost medium (OCM), designed to mimic the natural nutrient composition and microbial interactions. Compared to ZM, A. platensis H53 grown in OCM exhibited elevated growth by day 7. The specific growth rate in OCM was 0.20 day−1, higher than that of 0.17 day−1 in ZM, with optical density values reaching 1.57, compared to 1.13 in ZM. A 1.63-fold increase in biomass was observed in OCM, despite lower initial macronutrient concentrations. Nutrient use efficiency (NUE) in OCM was significantly improved, with nitrate (NO3−) and phosphate (PO43−) utilization up to 5.8-fold higher. Additionally, A. platensis H53 filaments in OCM were more tightly coiled, indicating a physiological change in response to lowered macronutrient concentrations. Microbial composition analysis using 16S rRNA gene amplicon sequencing revealed the presence of growth-promoting bacteria, including Pontibacter spp., Brevundimonas spp., and Aliihoeflea spp., likely contributing to nutrient cycling and enhanced growth. These findings suggest potential symbiotic interactions between cyanobacteria and non-cyanobacteria in the OCM system, promoting increased growth and productivity. This study is the first to propose such symbiosis in an extremely alkalophilic environment, offering another sustainable alternative to traditional chemical-based Arthrospira cultivation methods.

Diffuse interstellar bands as dust indicators: The contribution from 3D maps

Three-dimensional (3D) distributions of the 862 nm diffuse interstellar band (DIB) carrier have been computed based on Gaia parallaxes and DIB catalogues, in parallel with 3D maps of dust extinction density. Three-dimensional maps provide local diagnostics and information on the distribution of structures in addition to line-of-sight (LOS) integrated quantities, and allow us to focus on poorly studied low-extinction areas. They make cross-matching with other catalogues possible through estimates of the DIB and extinction along any given path. We re-examined the relationships between the density of DIB carriers and the absorption and emission properties of spatially co-located dust. Along with laboratory identifications of carriers, these properties may shed light on the formation and evolution of this organic matter. They may also help to model dust emission and absorption properties in a more detailed way. We used the 3D maps of 862 nm DIBs and of dust extinction, as well as available DIB equivalent width (EW) catalogues and published measurements of parameters characterizing the dust extinction law and the dust emission. We studied the relationships between the extinction-normalized 862 nm DIB EW and the extinction level, the total-to-selective extinction ratio $R_V$, and the dust far-IR emission spectral index beta . We re-visited the link between several DIBs and the UV absorption bump at 220 nm. The ratio of the 862 nm DIB carrier density to the optical extinction density (DIB$_ norm $) is increasing in low-density clouds, confirming with local values the trend seen in the LOS data. In both cases, the coefficients of a fitted power law fall within the range of those measured towards SDSS high-latitude targets for 20 different bands, ranking this DIB among those with a high increase, above that of the broad 4430 DIB. This is consistent with the recent measurement of a larger scale height above the Plane for the 862 nm DIB compared to that of the 4430 DIB. Using map-integrated 862 nm DIB EWs and extinctions along the paths to APOGEE targets with published proxies $R'_V$ for the total-to-selective extinction ratio, we found that, despite a large scatter, DIB$_ norm $ is positively correlated with $R'_V$ for those stars with low to moderate extinctions ($A_V$= 0.2 to 2--3 mag). Independently, using stars from the 862 nm DIB catalogue located outside the disk and for the same regime of extinction, DIB$_ norm $ is found to be globally anti-correlated with the Planck opacity spectral index beta . This is consistent with the observed anti-correlation between beta and $R'_ V $. In the light of a recent result on the variability of the carbon/silicate ratio in dust grains as a source of this anti-correlation, it suggests that DIB$_ norm $ increases with the fraction of carbonaceous to silicate grains in the co-located dust, in agreement with the carbonaceous nature of DIB carriers and recent evidences for a spatial correlation between DIB$_ norm $ and the fluxes of carbon-rich ejecta of asymptotic giant branch (AGB) stars. At higher extinction both trends disappear, and there is evidence for a trend reversal. Regarding the link between the height of the 220 nm UV absorption bump and extinction-normalized EWs of DIBs, we found that two factors explain the absence of previous clear results: the correlation disappears when we move from sigma -type to zeta -type DIBs and/or from single-cloud LOSs to paths crossing multiple clouds distant from each other; zeta -type bands can be used to predict low and high values of the bump height, provided one adds a correcting factor linked to the ambient radiation field (e.g. the 5780/5797 DIB ratio). We show examples of simple models of the bump height based on the 5780 band, the 5850 band and the 5780/5797 DIB ratio. We also found an anti-correlation between DIB$_ norm $ and the width of the bump, which similarly disappears from sigma -type to zeta -type DIBs. This suggests that a fraction of the bump is generated outside the dense molecular clouds. There are complex relationships between DIBs and dust; however, massive measurements of DIBs and extinction and the derived 3D maps may provide some constraints on the density, the nature, and the contribution to extinction and emission of the co-located dust. This requires large stellar spectroscopic surveys and space-based measurements of UV extinction.

Local H i absorption towards the magellanic cloud foreground using ASKAP

ABSTRACT We present the largest Galactic neutral hydrogen H i absorption survey to date, utilizing the Australian SKA Pathfinder Telescope at an unprecedented spatial resolution of 30 arcsec. This survey, GASKAP-H i, unbiasedly targets 2714 continuum background sources over 250 square degrees in the direction of the Magellanic Clouds, a significant increase compared to a total of 373 sources observed by previous Galactic absorption surveys across the entire Milky Way. We aim to investigate the physical properties of cold (CNM) and warm (WNM) neutral atomic gas in the Milky Way foreground, characterized by two prominent filaments at high Galactic latitudes (between $-45^{\circ }$ and $-25^{\circ }$). We detected strong H i absorption along 462 lines of sight above the 3$\sigma$ threshold, achieving an absorption detection rate of 17 per cent. GASKAP-H i’s unprecedented angular resolution allows for simultaneous absorption and emission measurements to sample almost the same gas clouds along a line of sight. A joint Gaussian decomposition is then applied to absorption-emission spectra to provide direct estimates of H i optical depths, temperatures, and column densities for the CNM and WNM components. The thermal properties of CNM components are consistent with those previously observed along a wide range of Solar neighbourhood environments, indicating that cold H i properties are widely prevalent throughout the local interstellar medium. Across our region of interest, CNM accounts for $\sim$30 per cent of the total H i gas, with the CNM fraction increasing with column density towards the two filaments. Our analysis reveals an anticorrelation between CNM temperature and its optical depth, which implies that CNM with lower optical depth leads to a higher temperature.

A-147 Comparative Precision of Platelet Counting by Optical, Fluorescent, and Impedance Techniques in Thrombocytopenia and Microcytosis Patients

Abstract Background In today's era of automation driven by Artificial Intelligence, even the most efficient automated hematology analyzer may struggle to accurately determine platelet count in patients with low platelet count and microcytosis. The Sysmex XN-series analyzer (Sysmex, Kobe, Japan) uses optical density, fluorescence, and electronic impedance methods for platelet counting. This study aimed to evaluate the accuracy of optical, fluorescence, and electrical impedance methods in estimating precise platelet counts in thrombocytopenic and microcytic patient samples. Methods A cross-sectional study was conducted in the Department of Laboratory Medicine at AIIMS, New Delhi, for 30 days consecutively. A total of 200 blood samples collected, of which 100 samples were of thrombocytopenia (<100x10^9/L) and 100 samples were of microcytosis (Mean corpuscular volume <76 femtoliters). MCV<76 fl was further categorized into 2 groups- MCV 60-70 fl and MCV 71-76 fl accordingly. Males and females were categorized and analyzed distinctly for thrombocytopenia and microcytosis. Statistical analysis was performed using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA) and the Bland Altman plot was analyzed. Results Of the 100 patients with thrombocytopenia (platelet<1 lakh/L) females were predominantly 54% while males were 46% and the median age was 29 years (0.02-70). Of the 100 patients with low MCV (<76 fl) females were 53%, while males were 47% and the median age was 29 years (0.3-69). The findings were compared against the standard manual platelet count method in peripheral smears verified by two expert pathologists. The Bland Altman plot for Platelet <1 lakh/L and MCV <76 fl were compared with standard manual platelet count method and the following findings were obtained i) In female with PLT- <1 lakh/L, PLT-O showed least bias (9.8) followed by PLT-F (10.4) and PLT-I (12.1) ii) In males with PLT-<1 lakh/L, PLT-O showed least bias(14.1) followed by PLT-F(16.5) and PLT-I(17.0) iii) For MCV 61-70fl, PLT-O showed the least bias (4.4) followed by PLT-F (6.7) and PLT-I (28.5) iv)For MCV 71-76fl, PLT-F showed least bias (-0.2) followed by PLT-O (1.5) and PLT-I(31.2). Conclusions Platelet count by optical method (PLT-O) detects platelet count more accurately with minimum bias when compared with PLT-F and PLT-I in patients with thrombocytopenia. In patients with microcytosis, PLT-I is less reliable in comparison to PLT-O and PLT-F. A limitation of the automated hematology analyzer is the inability to detect platelet clumps which is appreciated in peripheral smears.

A-166 Performance verification of a polyclonal anti-PF4 immunoassay in evaluation of Heparin Induced Thrombocytopenia (HIT)

Abstract Background Heparin-induced thrombocytopenia (HIT) is a severe complication of exposure to heparin that occurs in a small percentage of patients in anticoagulation therapy. HIT could happen regardless of the dose, exposure, schedule, or route of administration. HIT results from an autoantibody directed against endogenous platelet factor 4 (PF4) that forms complex with heparin. This antibody activates platelets and can cause life threatening arterial and venous thrombosis. Untreated HIT has a mortality rate as high as 20%. However, with appropriate screening, diagnosis and early intervention, mortality rates have been reported to be below 2%. The laboratory evidence in evaluating patients for HIT is crucial for early intervention. There are several assays available to diagnose Heparin Induced Thrombocytopenia (i.e., immunoassay and/or functional assay for HIT antibodies; serotonin release assays). The purpose of this study is to establish the measurement criteria and verify performance characteristics of a polyclonal anti-PF4 immunoassay. The test of interest (anti-PF4 immunoassay) is intended for the qualitative detection of the anti-heparin-platelet factor 4 antibodies in plasma or serum by ELISA. Methods The ASSERACHROM Assay (Diagnostica Stago, Inc. USA) was implemented in the automated Dynex DS2 ELISA Processing System (Dynex Technologies Inc USA). Two Dynex DS2 instrument comparisons along with precision, accuracy and measurement range for qualitative positive and negative results were studied. The reference method comparisons with serotonin release assay and other monoclonal anti-PF4 immunoassays were performed. To evaluate performance of the assay, we conducted precision, accuracy, and qualitative method comparison. We compared the assay of interest with two reference lab immunoassays and the gold standard reference method (serotonin release assay). The statistical analysis was carried out in EP Evaluator and GraphPad Prism. Results The negative control precision demonstrated CV of 9.2% (N=20, mean= 0.11, SD= 0.01). The analyte measurement is expressed as optical density (OD). The positive control precision characteristics showed CV of 8.1% (N=20, mean= 1.62, SD= 0.13). The threshold was defined as reference reagent absorbance (R6OD) times the variance factor divided by 100 ((R6OD* variance factor)/100= threshold (X)). This is used for positive (>the X) and negative (< the X) absorbance cut off for patient samples. The R6 reference material precision study resulted in a CV of 7.95% (N=20, mean= 2.1, SD= 0.17). The R6 variance factor per precision statistics at 3SD level was 23.85%. This is comparable to peer group variance provided in the assay package insert (23.5%). The polyclonal anti-PF4 immunoassay versus a reference monoclonal method (N=40) demonstrated agreement of 90.9% with positive agreement of 100% and negative agreement of 89.5%. When compared with gold standard serotonin release assay (N=40), the assay demonstrated agreement of 75% with positive agreement of 90% and negative agreement of 60%. The two instruments demonstrated agreement of 97.5% with positive agreement of 100% and negative agreement of 93.3% (Cohen's Kappa 94.6%; with Kappa > 75% indicates “high” agreement). Conclusions This anti-PF4 immunoassay is precise, accurate and comparable with three different reference methods. The test is best utilized when 4T score based clinical algorithm is used for evaluation of HIT.

Developing microalgae harvesting: Enhanced efficiency with magnetite-urea nanocomposites for sustainable astaxanthin biorefinery

Harvesting costs, accounting for 15–30 % of total expenses, are a major bottleneck in microalgae biorefinery due to low cell density and energy-intensive processes. This study investigates Fe3O4-Urea (Fe@urea) nanocomposites (NCs) to replace conventional methods and improve the harvesting efficiency (HE) of Chlorella zofingiensis, known for its high lipid and astaxanthin content. Urea-coated magnetite nanoparticles (NPs) with additional functional groups significantly improved harvesting performance across all pH levels compared to conventional Fe3O4 NPs. HE was evaluated using both optical density and cell counting methods, with the latter proving superior results by eliminating interference from unreacted salts and debris. Optimization of nanocomposite concentrations and culture pH with Fe@urea achieves 95 % HE at 800 mg L−1 and pH 3–4. The Langmuir and Freundlich models confirmed the nanocomposite's multifaceted capabilities. Characterization techniques included SEM, FTIR, EDX, zeta potential analysis, and TEM. This innovative approach can revolutionize large-scale microalgae biorefineries, enhancing sustainable biofuel and bioproduct production and supporting the Sustainable Development Goal of Affordable and Clean Energy (SDGs 7).

B-080 Development and Validation of a GQ1b IgG ELISA as a Diagnostic Aid for Subacute Demyelinating Polyneuropathies

Abstract Background Autoimmunity targeting GQ1b is associated with group of disorders that includes Miller-Fisher Syndrome (MFS), Bickerstaff Brainstem Encephalitis (BBE) and classic Guillain Barre Syndrome (GBS) with ophthalmoplegia. Collectively these are referred to as GQ1B-IgG related syndromes. The prevalence of GQ1b-IgG in this population of patients is high and has been reported to be >80% in well-defined clinical cohorts. Clinical phenotypic presentation is diverse and requires highly accurate antibody testing. The objective of this study was to evaluate a newly developed custom GQ1b-IgG assay, using a semi-quantitative ELISA-based method. Methods A GQ1b-IgG ELISA was developed and validated. Patient sera along with assay controls and a single-point calibrator are diluted and incubated in duplicate wells in a 96-well plate coated with GQ1b antigen. The plate is washed and incubated with anti-human-IgG secondary antibody conjugated with HRP. After a colorimetric reaction, optical densities (OD) are read. The OD of patient samples are compared against the calibrator, generating a cut-off index (COI). Samples ≥1.0 COI are considered positive. During the validation phase, analytical and clinical performance characteristics were evaluated. Studies included assessment of imprecision at multiple concentrations of GQ1b-IgG, accuracy using method comparison, analytical sensitivity and specificity, reference range, common interferences, plate drift, and clinical performance in relevant disease and control cohorts. Results Intra-assay imprecision (coefficient of variation; %CV) ranged from 1.0 - 16% across all concentrations of GQ1b-IgG. Inter-assay imprecision (%CV) ranged from 12.4 - 44.5% but was <20% at or above the assay cut-off. We found perfect qualitative agreement with an independent ELISA reference method (20 positive and 20 negative samples selected based on reference method results [reference interval <1:100]). Limit of detection (LOD) was calculated at a COI of 0.22. To assess analytical specificity, 40 samples with high concentrations of immunoglobulins (hypergammaglobulinemia [N=15], IgG M-protein [N=15] and systemic lupus erythematosus [N=10] were tested and all were negative. A reference interval study was performed on 140 healthy donor serums, all were negative. Interference studies were conducted on 2 positive and 2 negative serum samples, spiked for highly hemolytic (1000 mg/dL), lipemic (2000 mg/dL) and icteric (60 mg/dL) conditions. There was no impact on the assay even with high levels of a given interferent. Plate drift on 2 distinct patient pools tested across the plate (COI range 0.7 - 1.3) had overall plate CV’s ≤10%. ROC analysis was performed on a cohort of 278 patient samples (15 MFS cases and 263 controls [suspected demyelinating neuropathy or disease mimics]). A cut-off value of 1.0 (COI) was selected based on the Youden index. At this cut-off, clinical sensitivity for MFS was 87% while clinical specificity was 99.2%. Conclusions The observed performance of the GQ1b-IgG ELISA was acceptable for clinical laboratory implementation. A COI of ≥ 1.0 was selected for clinical use.

IgG reactivity to different desmoglein-3 ectodomains in pemphigus vulgaris: novel panels for assessing disease severity.

Pemphigus vulgaris (PV) is an autoimmune disease characterized by IgG autoantibodies targeting desmoglein-3 (Dsg3), leading to blistering of mucous membranes and skin. Although commercial ELISA kits effectively diagnose PV, correlation with clinical phenotype remains unclear. This study assesses multiple panels for monitoring disease severity and activity by profiling IgG autoantibodies against Dsg3's various extracellular ectodomains. We designed and expressed different extracellular domains of Dsg3 in HEK293T cell line and developed 15 different ELISA panels, each using a single or multi ectodomains encompassing the entire extracellular region of Dsg3 to detect specific autoantibodies against the particular part of Dsg3. To validate our approach, we compared our ELISA panel for the full Dsg3 (EC1-5) against a commercial kit using 154 random serum samples from PV patients, demonstrating a strong correlation. For evaluation of IgG autoantibody profiles in our panels, 59 PV patients were included, along with 11 bullous pemphigoid patients, and 49 healthy controls. For all the included subjects, 15 predefined ELISA panels were tested. The IgG autoantibodies against EC1 were detected in 86% of patients with a positive full Dsg3 ectodomain (EC1-5) ELISA, with 26% against EC2, 14% for EC3, 29% for EC4, and 23% for EC5. Among the panels with multiple Dsg3 ectodomains, EC1-3 and EC1-4 were representative of the entire Dsg3 ectodomain in terms of ELISA positivity across all included patients. A significant correlation (P<0.05) was observed between ELISA optical density (OD) and Pemphigus Disease Area Index (PDAI) scores in five panels, EC1, EC2-3, EC2-5, and EC3-4 in addition to the full ectodomain. It suggests an association with disease severity. Interestingly, while the ELISA panel for the entire Dsg3 extracellular ectodomains did not differentiate disease phases, in three of our panels, including EC1, EC3-5, and EC2-5, ANOVA analysis showed a statistically significant difference between the groups of patients in remission, partial remission or persistent lesions, and those with active disease (new cases or relapse). Among these three panels, EC1 was the only one that showed a significant difference in the multiple comparisons analysis; patients in the active phase had higher levels of autoantibodies than those in 'partial remission or persistent lesions' and 'complete remission' groups. The level of autoantibodies against EC1 was not only correlated with the full ectodomain but also associated with higher disease severity and active disease phase. This study indicates that a detailed autoantibody profile against Dsg3 ectodomains could serve as a marker for PV severity and activity which may potentially enhance early treatment initiation.

Characterization of the immunodominant regions of Senecavirus A-VP1 structural protein via ELISA epitope mapping

Senecavirus A (SVA) is an RNA virus in the family Picornaviridae that has been detected in swine-production systems and is associated with vesicular disease and neonate mortality. The viral capsid is composed of four structural proteins: VP1–VP4. Although the VP1 protein has been reported to be the most immunogenic protein in vivo, no information on the immunodominant regions of the SVA polyprotein is available. The objective of this study was to identify the immunodominant regions of SVA polyprotein using an enzyme-linked immunosorbent assay (ELISA) epitope-mapping approach. The binding effect of SVA polyclonal antibody (SVA-pAb), SVA-VP1 monoclonal antibodies (SVA-mAb), and SVA-positive sera from clinically affected animals were characterized using a set of 18 overlapping SVA VP1-derived peptides by indirect and blocking ELISAs. All VP1 peptides yielded significant signal against SVA-pAb and SVA-VP1-mAb upon indirect ELISA. One peptide (aa 1–20) showed significantly high optical density on SVA recombinant VP1 protein (rVP1) and whole-virus-based indirect ELISAs. The blocking ELISA results demonstrated that peptides spanning aa 165–185 and 225–245 had a 50 % or greater inhibitory effect on SVA-pAb, while six groups of overlapping peptides spanning aa 1–35, 45–80, 90–140, 150–170, 195–230, and 240–264 and two groups of overlapping peptides spanning aa 1–50 and 60–264 showed a 50 % inhibitory effect or greater on swine VP1-mAb and SVA-seropositive swine serum, respectively, against SVA rVP1. Three-dimensional protein homology modeling showed that the peptides binding SVA-pAb are located on the outer surface of the viral capsid, while SVA mAbs and swine-positive sere can bind to epitopes located in both the inner and outer surfaces of the capsid. These linear epitopes showed differential binding and inhibitory activity on mAb and pAb; however, further studies will be necessary to evaluate whether they can act as decoy or neutralizing epitopes. Because mAb antibodies demonstrated a high binding affinity for this set of peptides, this information could lay the foundation for generating and screening specific antibodies for therapeutic potential.

ELISA-R: an R-based method for robust ELISA data analysis.

Enzyme-linked immunosorbent assay (ELISA) is a technique to detect the presence of an antigen or antibody in a sample. ELISA is a simple and cost-effective method that has been used for evaluating vaccine efficacy by detecting the presence of antibodies against viral/bacterial antigens and diagnosis of disease stages. Traditional ELISA data analysis utilizes a standard curve of known analyte, and the concentration of the unknown sample is determined by comparing its observed optical density against the standard curve. However, in the case of vaccine research for complicated bacteria such as Mycobacterium tuberculosis (Mtb), there is no prior information regarding the antigen against which high-affinity antibodies are generated and therefore plotting a standard curve is not feasible. Consequently, the analysis of ELISA data in this instance is based on a comparison between vaccinated and unvaccinated groups. However, to the best of our knowledge, no robust data analysis method exists for "non-standard curve" ELISA. In this paper, we provide a straightforward R-based ELISA data analysis method with open access that incorporates end-point titer determination and curve-fitting models. Our modified method allows for direct measurement data input from the instrument, cleaning and arranging the dataset in the required format, and preparing the final report with calculations while leaving the raw data file unchanged. As an illustration of our method, we provide an example from our published data in which we successfully used our method to compare anti-Mtb antibodies in vaccinated vs non-vaccinated mice.

A-232 Characterize and reduce positive interference of hemolysis on Fungitell testing for (1, 3)-β-glucan (BDG) in serum

Abstract Background Fungitell is an FDA cleared in vitro diagnostic product with an indication for use as an adjunct test for the diagnosis of invasive fungal infection. This assay has been established using a kinetic photometric method. A key aspect of this (1→3)-β-glucan (BDG) activated cascade reaction is its characteristic kinetic optical density profile. Interference which affects the BDG kinetic profile makes data analysis and interpretation difficult. This interference is roughly classified as being associated with hemolysis, icterus, lipemia (collectively HIL), and other non-specific factors. Methods In this study, we found that the use of 5% PEG-8000 supplemented serum (1:1 ratio of the volume of serum to 10% PEG-8000 solution with 0.1 M Tris-Cl, pH 7.4), heated at 80°C for 3.5 min, followed by centrifugation at 15,000 x g for 10 minutes (PEG-X Method) can largely eliminate the hemolysis-associated interference. Results The treated serum Fungitell BDG reaction curves are more like the ideal BDG kinetic profile. The recovery of introduced β-glucan has been shown to be acceptable (80-120%) for hemolyzed specimens. Conclusions This simple and rapid procedure shows promise as a pre-analytical treatment to reduce serum interference in the Fungitell β-glucan test for a substantial fraction of samples that produce currently unreportable results.

Potential utility of RSAD2 transcript and protein in early detection of pregnancy in buffaloes.

This study investigates a novel early pregnancy marker in water buffaloes, focusing on RSAD2 mRNA expression, known to be upregulated by interferon-tau (IFNT) during pregnancy. While RSAD2 is primarily recognized for its antiviral effect, we hypothesized its role as a conceptus-induced component in regulating pregnancy in buffaloes. Given its differential expression compared to other IFNT-induced genes in cows, RSAD2 may serve as a biomarker for early pregnancy detection in buffaloes. RNA, cDNA, and plasma samples were obtained from archived samples collected before insemination (d0) and at d20, d25 and d40 after insemination. Twelve RNA samples, having optimal optical density and concentration, from six pregnant and six non-pregnant buffaloes were selected. The cDNA was analyzed to measure the abundance of RSAD2 mRNA using real-time quantitative PCR (RT-qPCR) and plasma for protein expression analysis using Western blot. The RT-qPCR analysis showed a transcript of RSAD2 increased significantly by 7-fold and 6-fold on d20 and d25, compared to both d0 and d40 in the pregnant group only. At d20, the sensitivity of RSAD2 was 100% and the specificity was 83.3%, and at d25-d both the sensitivity and specificity was 100%, indicating low incidences of misdiagnosing early pregnancy in buffaloes. In the non-pregnant group, RSAD2 expression remained low and did not change after insemination. Western blot analysis revealed an immunoreactive RSAD2 protein band. Densitometry analysis of the RSAD2-specific protein band, based on gray mean value, showed significantly increased expression of RSAD2 at d25 compared to d0 in the pregnant group. In conclusion, these results indicated that RSAD2 expressions at both the mRNA and protein levels show promising potential for detecting pregnancy at d25 post-insemination.

Изменение бактериального фитнеса в ходе экспериментальной адаптации Pseudomonas aeruginosa к колистину

Pseudomonas aeruginosa, the opportunistic pathogen, occupies one of the leading places in the structure of pathogens causing nosocomial infections, which is due to high adaptive potential and the ability to quickly develop antimicrobial resistance. The study aimed to assess the influence of the P. aeruginosa adaptation to colistin on bacterial fitness. A total of nine isolates obtained during the experimental evolution of the P. aeruginosa strain (laboratory number 1202) under conditions of increasing colistin concentrations, the growth kinetics of which was compared to that of wild type strain, were included in the study; the whole genome sequencing of all isolates was performed, and the minimum inhibitory concentration of colistin was determined. Growth rate was estimated using the Varioskan LUX multimodal reader (Thermo Scientific, USA) throughout 18 h at 37 °С; optical density (OD) at λ = 600 nm was measured every 15 min. The maximum growth rate (GRmax, i.e. the maximum change in OD within 1h) and the time to reach 50% of the maximum OD reported when growing the wild type Ра_1202_0 strain (T_OD50%) were considered. Isolates of the clone carrying mutations of the genes phoQ, lptA, and prs showed low fitness values compared to wild type strains. For example, GRmax of the isolate Ра_1202_43 was 0.029 OD/h vs. 0.182 OD/h reported for the original isolate Ра_1202_0, and it reached OD50% 4.6 h later. The growth characteristics of the clone carrying mutations of lpxL and lptB, as well as the clone carrying mutant pmrB were generally comparable with the characteristics of the wild type strain. Thus, the genome modifications observed during the P. aeruginosa adaptation to colistin have an ambiguous effect on bacterial fitness.

Application of a Novel Disposable Flow Cell for Spectroscopic Bioprocess Monitoring

The evaluation of the analytical capabilities of a novel disposable flow cell for spectroscopic bioprocess monitoring is presented. The flow cell is presterilized and can be connected to any kind of bioreactor by weldable tube connections. It is clamped into a reusable holder, which is equipped with SMA-terminated optical fibers or an integrated light source and detection unit. This modular construction enables spectroscopic techniques like UV-Vis spectroscopy or turbidity measurements by scattered light for modern disposable bioreactors. A NIR scattering module was used for biomass monitoring in different cultivations. A high-cell-density fed-batch cultivation with Komagataella phaffii and a continuous perfusion cultivation with a CHO DG44 cell line were conducted. A high correlation between the sensor signal and biomass or viable cell count was observed. Furthermore, the sensor shows high sensitivity during low turbidity states, as well as a high dynamic range to monitor high turbidity values without saturation effects. In addition to upstream processing, the sensor system was used to monitor the purification process of a monoclonal antibody. The absorption module enables simple and cost-efficient monitoring of downstream processing and quality control measurements. Recorded absorption spectra can be used for antibody aggregate detection, due to an increase in overall optical density.

The molecular mechanisms by which the NLRP3 inflammasome regulates blood-brain barrier permeability following cryptococcal meningitis

ObjectiveTo investigate the mechanism underlying the regulation of blood-brain barrier permeability changes during cryptococcal meningitis by NLRP3 and Vimentin. MethodsSprague-Dawley rats were treated with WT Cryptococcus neoformans (Cn) or CPS1-/- Cn. Neuronal apoptosis was assessed using TUNEL staining, and pathological changes were observed using electron microscopy and HE staining. The expressions of NLRP3, Vimentin, and NF-κB in the cerebral cortex and human brain microvascular endothelial cells (HBMECs) were examined through Western blot and qRT-PCR. siNLRP3 and siVimentin were separately transfected into HBMECs, the expressions of specific factors were assessed. NF-κB and Vimentin levels were detected through immunofluorescence, apoptosis was measured using flow cytometry, and changes in the optical density (OD) of HRP were determined using ELISA. ResultsThe expressions of NLRP3, Vimentin, and NF-κB were upregulated following intervention with WT Cn in vivo and in vitro. Electron microscopy revealed loose nuclear membranes in neurons and increased apoptosis in the cerebral cortex and hippocampus induced by WT Cn, accompanied by a reduction in the OD of HRP in vitro. siNLRP3 decreased the expressions of Vimentin, nuclear NF-κB, and β-Tubulin in HBMECs, while siVimentin downregulated total NLRP3 and nuclear NF-κB levels. Both siNLRP3 and siVimentin reduced cell apoptosis after WT Cn infection. HBMECs displayed a reduced monolayer permeability to HRP and improved cell structure arrangement. ConclusionVimentin and the NLRP3 inflammasome are both implicated in the pathological process of cryptococcal meningitis. An interaction between Vimentin and the NLRP3 inflammasome is evident, likely mediated through the NF-κB signaling pathway.