Fluorescence Units Research Articles

Silver nanoparticles incorporated dental restorative resin and its antibiofilm effect.

Dental restoration materials are susceptible to bacterial biofilm formation, which damages the restorations and causes oral health problems. Therefore, to overcome this, silver nanoparticles (AgNPs) are studied widely due to their antimicrobial, anti-inflammatory and healing properties. The purpose of this study was to develop a strategy for incorporating AgNPs onto the surface of bisacrylic resin (Bis) to evaluate its antibiofilm effects using Streptococcus sanguinis and Actinomyces naeslundii. AgNPs with an average size of 25 nm at two different concentrations were dispersed on the Bis surface (Bis-AgNPs) by mechanical deposition. Ag release was quantified until 7 days of incubation. Bacterial growth was assessed using a viability assay kit and observed using confocal microscopy. The biofilm biomass was quantified using arbitrary fluorescence units. Cell viability was evaluated using an MTT assay. The results showed that Bis-AgNPs significantly inhibited biofilm formation along with a significant difference in the viability of human gingival fibroblasts. The quantification confirmed a decrease in Ag release over time, and elemental mapping showed AgNP penetration up to 10 µm from the surface. Therefore, it was concluded that Bis-AgNPs presented enhanced antibiofilm properties, even at a concentration with no adverse effects. Therefore, this nanocomposite may be a promising alternative for biofilm control in temporary restorative materials.

In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism

BackgroundConservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24–48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media. Material and methodsProcessed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca2+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca2+ were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population. ResultsAfter 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca2+ increased in all treatment groups compared to time 0h. Discussion and conclusionViable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.

Anti-angiogenic and antioxidant effects of axitinib in human retinal endothelial cells: implications in diabetic retinopathy.

Diabetic retinopathy is a secondary microvascular complication of diabetes mellitus. This disease progresses from two stages, non-proliferative and proliferative diabetic retinopathy, the latter characterized by retinal abnormal angiogenesis. Pharmacological management of retinal angiogenesis employs expensive and invasive intravitreal injections of biologic drugs (anti-vascular endothelial growth factor agents). To search small molecules able to act as anti-angiogenic agents, we focused our study on axitinib, which is a tyrosine kinase inhibitor and represents the second line treatment for renal cell carcinoma. Axitinib is an inhibitor of vascular endothelial growth factor receptors, and among the others tyrosine kinase inhibitors (sunitinib and sorafenib) is the most selective towards vascular endothelial growth factor receptors 1 and 2. Besides the well-known anti-angiogenic and immune-modulatory functions, we hereby explored the polypharmacological profile of axitinib, through a bioinformatic/molecular modeling approach and in vitro models of diabetic retinopathy. We showed the anti-angiogenic activity of axitinib in two different in vitro models of diabetic retinopathy, by challenging retinal endothelial cells with high glucose concentration (fluctuating and non-fluctuating). We found that axitinib, along with inhibition of vascular endothelial growth factor receptors 1 (1.82 ± 0.10; 0.54 ± 0.13, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) and vascular endothelial growth factor receptors 2 (2.38 ± 0.21; 0.98 ± 0.20, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively), was able to significantly reduce (p < 0.05) the expression of Nrf2 (1.43 ± 0.04; 0.85 ± 0.01, protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in retinal endothelial cells exposed to high glucose, through predicted Keap1 interaction and activation of melanocortin receptor 1. Furthermore, axitinib treatment significantly (p < 0.05) decreased reactive oxygen species production (0.90 ± 0.10; 0.44 ± 0.06, fluorescence units in high glucose vs . axitinib 1 µM, respectively) and inhibited ERK pathway (1.64 ± 0.09; 0.73 ± 0.06, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in HRECs exposed to high glucose. The obtained results about the emerging polypharmacological profile support the hypothesis that axitinib could be a valid candidate to handle diabetic retinopathy, with ancillary mechanisms of action.

Circularly Polarized Electroluminescence from Intrinsically Axial Chiral Materials Based on Bidibenzo[b,d]furan/bidibenzo[b,d]thiophene

AbstractHerein, by connecting chiral bidibenzo[b,d]furan/bidibenzo[b,d]thiophene and multi‐resonance thermally activated delayed fluorescence (MR‐TADF) units, two pairs of intrinsically axial chiral MR‐TADF materials (R/S‐BDBF‐BNO and R/S‐BDBT‐BNO) realize mirror‐symmetric circularly polarized luminescence (CPL) and narrowband emission. The chiral moieties effectively engage in frontier molecular orbital distributions through the establishment of covalent bonds with boron atoms. The proportions of (1,1′‐bidibenzo[b,d]furan)‐2,2′‐diol in the highest occupied and lowest unoccupied natural transition orbitals (HONTO/LUNTO) are 25.3% and 64.7%, respectively. In addition, BDBT‐BNO processes a dual‐channel transition mode, thus the (1,1′‐bidibenzo[b,d]thiophene)‐2,2′‐diol constitutes 22.8%, 24.3% and 54.0%, 46.9% of the distributions of HONTO, HONTO‐1 and LUNTO, LUNTO+1, respectively. The high proportions endow R/S‐BDBF‐BNO and R/S‐BDBT‐BNO with good CPL properties with dissymmetry factors (|gPL|) of 1.7/1.8 × 10−3. Correspondingly, the circularly polarized organic light‐emitting diodes based on the enantiomers exhibit mirror symmetrical circularly polarized electroluminescence with |gEL| factors of 1.5/1.6 × 10−3. Furthermore, the introduction of sulphur atoms enables BDBT‐BNO to have a high maximum external quantum efficiency of 35.7% in device.

Comparison of CellRox green fluorescence upon thawing on in vitro Bos taurus and Bos indicus embryos cryopreserved by slow freezing or vitrification.

The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

Ultrasensitive Fluorescent Microsensors Based on Aptamers Modified with SYBR Green I for Visual Quantitative Detection of Organophosphate Pesticides.

Organophosphate pesticides (OPs) are widely utilized in agricultural production, and the residues threaten public health and environmental safety due to their toxicity. Herein, a novel and simple DNA aptamer-based sensor has been fabricated for the rapid, visual, and quantitative detection of profenofos and isocarbophos. The proposed DNA aptamers with a G-quadruplex spatial structure could be recognized by SYBR Green I (SG-I), resulting in strong green fluorescence emitted by SG-I. The DNA aptamers exhibit a higher specific binding ability to target OP molecules through aromatic ring stacking, disrupting the interaction between SG-I and DNA aptamers to induce green fluorescence quenching. Meanwhile, the fluorescence wavelength of G-quadruplex fluorescence emission peaks changes, accompanied by an obvious fluorescence variation from green to blue. SG-I-modified aptasensor without any additive reference fluorescence units for use in multicolor fluorescence assay for selective monitoring of OPs was first developed. The developed aptasensor provides a favorable linear range from 0 to 200 nM, with a low detection limit of 2.48 and 3.01 nM for profenofos and isocarbophos, respectively. Moreover, it offers high selectivity and stability in real sample detection with high recoveries. Then, a self-designed portable smartphone sensing platform was successfully used for quantitative result outputs, demonstrating experience in designing a neotype sensing strategy for point-of-care pesticide monitoring.

#2395 Elevated circulating ACE2 activity is related with a major COVID-19 severity

Abstract Background and Aims The angiotensin 2 converser enzyme (ACE2) is the cell entry receptor for SARS-CoV-2. SARS-CoV2 participates in ACE2 shedding from cell membrane, increasing soluble ACE2. The present study aims to evaluate if ACE2 serum levels are to COVID-19 prognosis. Method During the first wave of COVID-19 pandemic and during March and April 2020, 2118 first emergency attendance serum samples from diagnosed COVID-19 patients from Barcelona Vall d'Hebron Hospital were obtained. Age, sex, previous comorbidities, clinical and analytical variables were collected from the patient clinical records. Patients that were admitted to ICU, required mechanical ventilation or died due to COVID-19 were considered severe patients. Serum ACE2 activity was measured by enzymatic activity assay. Results Patients with severe COVID-19 were older and with a major number of comorbidities (Table 1). Circulating ACE2 activity at admission was increased in 0.34 relative fluorescence units (RFUs)/ng/μL (CI 95%: 0.24-0.43, p &amp;lt; 0.001) in patients that developed severe COVID-19. A logistic regression model adjusted by age, sex, diabetes, hypertension, obesity, cardiovascular disease, chronic kidney disease and renin angiotensin blockers, showed that high circulating ACE2 activity was still related to a worse prognosis (OR: 2.0, 95% CI: 1.7–2.4, p &amp;lt; 0.001). High serum ACE2 activity ≥0.56 RFU/ng/μL provided the best sensitivity (62%) and specificity (61%) relation, whereas values ≥1.3 RFU/ng/μL were significantly related to a worse prognosis of the disease with a 95% specificity. Conclusion High levels of circulating ACE2 activity when patients with COVID-19 are admitted to the Emergency Department are independently related with a subsequent severe disease. Additionally, although most of the comorbidities analyzed are significantly increased in severe COVID-19 patients as compared to mild COVID-19 patients, the results of the logistic regression model point out that they seem not to be a risk factor in the overall model, except for obesity, age and sex.

Real-time chemostratigraphy at wellsite; removing drilling uncertainties

Real-time chemostratigraphy is a workflow deployed at wellsites to remove stratigraphic uncertainty and support drilling operations. This study presents the chemostratigraphic wellsite workflow which, during drilling operations, support either the placement of casing points (using predefined geochemical markers to target a depth above a specific stratigraphic feature) or aid geosteering. Geosteering is typically employed during the drilling of a horizontal well and is done to keep the drill within a certain zone or horizon to maximise production. Wellsite chemostratigraphy has been effectively deployed to wellsites within multiple basins around the globe and has recently been utilised in the Perth Basin. The wellsite workflow starts before the deployment, with offset wells analysed by inductively coupled plasma (ICP) spectroscopy within a laboratory-based setting. This builds the correlative element-based framework and identifies the trends required to assist with either core placement, casing point, or geosteering. Samples are analysed by energy dispersive x-ray fluorescence units (these data are then used to confirm the zone previously defined) and can be identified at wellsite using the onsite tool, the selected elements of which are those less likely to be affected by drilling mud contamination and loss of circulation material (LCM). However, during drilling operation, there may be instances where LCM must be added to the drilling muds, which may have an effect on the data quality for certain elements. With a dedicated ICP spectrometry elemental database behind the wellsite operation, machine learning tools may be employed to ‘repair’ correlation critical elements.

Comparison of the Applied Biosystems® SeqStudio™ and 3500 Genetic Analyzers

ABSTRACT We report on a comparison of the performance of the Applied Biosystems® SeqStudioTM Genetic Analyzer with the 3500 Genetic Analyzer when analysing trace DNA samples. Totally, 162 profiles generated from ‘trace DNA’ were analysed on both instruments with comparable number of alleles detected, relative fluorescence units (RFU) and likelihood ratio (LR) values. The SeqStudioTM was found to be suitable for the analysis of trace DNA samples.

A novel DNase assay reveals low DNase activity in severe asthma.

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 (n = 439) and from healthy controls (n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation.NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways.

Medicinal plant extracts interfere in gastric cancer stem cells fluorescence-based assays

Fluorescence is used in various biological assays due to its high sensitivity, versatility, and precision. In recent years, studies using medicinal plant extracts have increased. However, fluorescence-based assays could be biased by plant metabolites autofluorescence. To address this issue, this study investigated the interference caused by methanolic extracts and chloroform fractions of three medicinal plants in three fluorescence-based assays on gastric cancer stem cells(CSC): resazurin reduction, confocal microscopy, and flow cytometry. CSC were isolated based on CD44 surface marker, incubated with methanolic extracts and chloroform fractions of Buddleja incana, Dracontium spruceanum, Piper aduncum. Resazurin assay evidenced that CSC exposed to extracts and fractions from the three plants showed significant differences in relative fluorescence units (RFU) levels (p < 0.001) compared to the unexposed groups after a 3-hour incubation. In addition, DMSO-treated CSC exposed to extracts and fractions had significantly lower fluorescence levels than living ones, but higher than extracts and fractions without cells. In confocal microscopy, cancer stem cells exposed to extracts and fractions of B. incana and P. aduncum were observed in the same emission spectra of the CSC markers. In flow cytometry, CSC exposed to extracts and fractions without any fluorescent dyes were detected in the double positive quadrants for CSC markers (CD44+/CD133 + ). Among the three plants, D. spruceanum exhibited the least interference. These results show that methanolic extracts and chloroform fractions contain autofluorescent metabolites that interfere with fluorescence-based assays. These results highlight the importance of a prior evaluation for possible fluorescence interference to avoid interpretation biases in fluorescence assays.

BSA nanoclusters-based sensor for detection of dopamine in schizophrenia from biofluids

Objective To develop nontoxic and stable fluorescent emission B-Cu nanoclusters (NCs) for the specific detection of dopamine at low concentrations in cerebrospinal fluid (CSF). Significance Fluorescent gold and copper NCs conjugated with proteins, such as bovine serum albumin (BSA), offer photostability and healthcare potential. This study focused on fabricating B-Cu NCs that exhibited superior characteristics for sensitive dopamine detection. Methods The study employed various instrumental techniques including attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), spectrofluorometry, and transmission electron microscopy (TEM) to characterize the formulated B-Cu NCs. The NCs were synthesized, resulting in particle size ∼300 nm. The highest observed fluorescence was recorded at 24542.81 relative fluorescence units (RFU). Results The introduction of dopamine at concentrations of 0.1, 0.2, 0.3, and 0.4 ng/mL led to decreased fluorescence in both B-Au and B-Cu NCs due to an electron transport system. This reduction in fluorescence allowed dopamine concentration analysis in phosphate buffer and biological fluids such as blood plasma and CSF. B-Cu NCs showed potential as a biosensing system for point-of-care (POC) applications, specifically for diagnosing schizophrenia. Conclusion The study successfully synthesized stable and nontoxic B-Cu NCs with enhanced fluorescent emission properties. These NCs exhibited the capacity to detect dopamine at low concentrations in CSF. The study’s findings hold promise for future applications, particularly in the development of a B-Cu NCs-based biosensing system for convenient POC detection of schizophrenia by both patients and clinicians. The potential impact of this technology on healthcare and biomedical fields is substantial.

Solid Phase Oligo-DNA Extraction from Complex Medium Using an Aminated Graphene/Nitrocellulose Membrane Hybrid.

A hybrid material, consisting of commercially available nitrocellulose (NC) membrane non-covalently modified with amino-polyethylene glycol functionalized reduced graphene oxide (NH2-PEG-rGO) nanoparticles, was successfully synthesized for oligonucleotide extraction. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the modification of the NC membrane, revealing characteristic peaks of both compounds, i.e., NC and NH2-PEG-rGO. Scanning Electron Microscopy (SEM) exhibited morphological changes in the NC/NH2-PEG-rGO hybrid membrane, marked by the introduction of NH2-PEG-rGO particles, resulting in a distinctly smothered surface compared to the porous surface of the NC control membrane. Wettability assays revealed hydrophobic behavior for the NC/NH2-PEG-rGO hybrid membrane, with a water contact angle exceeding 90°, contrasting with the hydrophilic behavior characterized by a 16.7° contact angle in the NC membrane. The performance of the NC/NH2-PEG-rGO hybrid membrane was evaluated for the extraction of ssDNA with fewer than 50 nucleotides from solutions containing various ionic species (MnCl2, MgCl2, and MnCl2/MgCl2). The NC/NH2-PEG-rGO hybrid membrane exhibited optimal performance when incubated in MgCl2, presenting the highest fluorescence emission at 525 relative fluorescence units (r.f.u.). This corresponds to the extraction of approximately 610 pg (≈13%) of the total oligo-DNA, underscoring the efficacy of the pristine material, which extracts 286 pg (≈6%) of oligo-DNA in complex solutions.

Development of a CRISPR-Cas12a based assay for the detection of swine enteric coronaviruses in pig herds in China

Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine deltacoronavirus (PDCoV) and Swine acute diarrhea syndrome coronavirus (SADS-CoV) rank among the most frequently encountered swine enteric coronaviruses (SECoVs), leading to substantial economic losses to the swine industry. The availability of a rapid and highly sensitive detection method proves beneficial for the monitoring and surveillance of SECoVs. Based on the N genes of four distinct SECoVs, a novel detection method was developed in this study by combining recombinant enzyme polymerase isothermal amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) 12a. Results showed that the cut-off value of CRISPR-Cas12a assay for SADS-CoV, PEDV, PDCoV and TGEV was 2.19 × 104 Relative Fluorescence Units (RFU), 1.57 × 104 RFU, 3.07 × 104 RFU and 1.64 × 104 RFU, respectively. The coefficient of variation (CV) of within and between runs by CRISPR-Cas12a assay for 6 clinical diarrhea samples were both less than 10%. The CRISPR-Cas12a assay demonstrated high specificity for TGEV, PEDV, PDCoV, and SADS-CoV with no cross-reactivity to other common swine viruses. This method also exhibited a low limit of detection of 2 copies for each virus. Additionally, the results demonstrated a perfect agreement (100%) between the CRISPR-Cas12a assay and the RT-qPCR assay. Finally, a total of 494 pig samples from the field tested by CRISPR-Cas12a assay showed that positive rate for SADS-CoV, TGEV, PDCoV and PEDV was 0, 0, 1.2% and 48.6%, respectively. The results suggested the great potential of CRISPR-Cas12a assay to detect SECoVs in the field.

Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Effect of the use of bromelain associated with bioactive glass-ceramic on dentin/adhesive interface.

To evaluate the effect of bromelain associated with Biosilicate on the bond strength (BS) of a universal adhesive system to sound (SD) and caries-affected dentin (CAD), and on the proteolytic activity. Cavities were prepared in 360 molars, half submitted to cariogenic challenge. Teeth were separated into groups (n=20): Control-No treatment; CHX-0.12% chlorhexidine; NaOCl-5% sodium hypochlorite; Br5%-5% bromelain; Br10%-10% bromelain; Bio-10% Biosilicate; NaOClBio-NaOCl+Bio; Br5%Bio-Br5%+Bio; Br10%Bio-Br10%+Bio. Following treatments, the adhesive system was applied, and cavities were restored. Samples were sectioned into sticks and stored at 37 °C for 24 h, 6 months, and 1 year. Microtensile BS (2-way ANOVA, Bonferroni's test, α=0.05), fracture patterns (SEM), and adhesive interfaces (TEM) were evaluated. Bacterial collagenase assay and in situ zymography were performed. In CAD, Br10% presented higher BS (p=0.0208) than Br5%Bio. Br5% presented higher BS (p=0.0033) after 6 months than after 24 h; and association of treatments, higher BS (p<0.05) after aging than after 24 h. Mixed fractures were the most prevalent. Association of treatments promoted a more uniform hybrid layer with embedded Bio particles. Experimental groups presented lower (p<0.0001) relative fluorescence units than Control. Bromelain, associated or not with Bio, showed collagenolytic degradation. Bromelain associated with Biosilicate did not affect the BS to SD. In CAD, Br5%Bio decreased immediate BS but had no long-term influence. This association decreased the proteolytic activity. Bromelain and Biosilicatemay enhance the longevity of adhesive restorations by inhibiting endogenous proteases.

Fluorescence imaging to visualize the recurrent laryngeal nerve during thyroidectomy procedures: analysis of 65 cases and 81 nerves.

Recurrent laryngeal nerve (RLN) injury after thyroidectomy is relatively common. Locating the RLN prior to thyroid dissection is paramount to avoid injury. We developed a fluorescence imaging system that permits nerve autofluorescence. We aimed to determine the sensitivity and specificity of fluorescence imaging at detecting theRLN relative to thyroid and other background tissue and compared it to white light. In this prospective study, 65 patients underwent thyroidectomy from January to April 2022 (16 bilateral thyroid resections) using white and fluorescent light. Fluorescence intensity [relative fluorescence units (RFU)] was recorded for RLN, thyroid, and background. RFU mean, minimum, and maximum values were calculated using Image J software. Thirty randomly selected pairs of white and fluorescent light images were independently reviewed by two examiners to compare RLN detection rate, number of branches, and length and minimum width of nerves visualized. Parametric and nonparametric statistical analysis was performed. All 81 RNLs observed were visualized more clearly under fluorescence (mean intensity, µ = 134.3 RFU) than either thyroid (µ = 33.7, p < 0.001) or background (µ = 14.4, p < 0.001). Forest plots revealed no overlap between RLN intensity and that of either other tissue. Sensitivity and specificity for RLN were 100%. All 30 RLNs and all 45 nerve branches were clearly visualized under fluorescence, versus 17 and 22, respectively, with white light (both p < 0.001). Visible nerve length was 2.5 × as great with fluorescence as with white light (µ = 1.90 vs. 0.76cm, p < 0.001). In 65 patients and 81 nerves, RLN detection was markedly and consistently enhanced with autofluorescence neuro-imaging during thyroidectomy, with 100% sensitivity and specificity.

Atrial fibrillation in human patients is associated with increased collagen type V and TGFbeta1

Background and aimAtrial fibrosis is an important factor in initiating and maintaining atrial fibrillation (AF). Collagen V belongs to fibrillar collagens. There are, however no data on collagen V in AF. The aim of this work was to study the quantity of collagen V and its relationship with the number of fibroblasts and TGF- b 1 expression in patients in sinus rhythm (SR) and in patients with atrial fibrillation (AF). MethodsWe used quantitative immuhistochemistry to study collagen V in right and left atrial biopsies obtained from 35 patients in SR, 35 patients with paroxysmal AF (pAF) and 27 patients with chronic, long-standing persistent AF (cAF). In addition, we have quantified the number of vimentin-positive fibroblasts and expression levels of TGF-β1. ResultsCompared to patients in SR, collagen V was increased 1.8- and 3.1-fold in patients with pAF and cAF, respectively. In comparison with SR patients, the number of vimentin-positive cells increased significantly 1.46- and 1.8-fold in pAF and cAF patients, respectively.Compared to SR patients, expression levels of TGF-ß1, expressed as fluorescence units per tissue area, was significantly increased by 77 % and 300 % in patients with pAF and cAF, respectively. Similar to intensity measurements, the number of TGFß1-positive cells per 1 mm2 atrial tissue increased significantly from 35.5 ± 5.5 cells in SR patients to 61.9 ± 12.4 cells in pAF and 131.5 ± 23.5 cells in cAF. In both types of measurements, there was a statistically significant difference between pAF and cAF groups. ConclusionsThis is the first study to show that AF is associated with increased expression levels of collagen V and TGF-ß1indicating its role in the pathogenesis of atrial fibrosis. In addition, increases in collagen V correlate with increased number of fibroblasts and TGF-β1 and are more pronounced in cAF patients than those in pAF patients.

Heterozygous Variants in the Clpbgene Associated with Neutropenia Affect Neutrophil Function

Introduction The CLPB gene encodes a caseinolytic peptidase belonging to the ATPase family and acts as a mitochondrial chaperone. While biallelic mutations in CLPB are associated with the 3-methylglutaconic aciduria and severe neutropenia, (MIM number: 616271 3), some heterozygous variants seem to induce neutropenia exclusively. In the current study, we evaluated for the first time a function of neutrophils among neutropenic carriers of CLPB mutation. Methods Among 282 individuals with neutropenia studied with targeted Next-Generation Sequencing (panel of 725 genes related to hematological disorders), three neutropenic probands with heterozygous CLPB amino acid substitutions were identified. Neutrophils from patients and control healthy donors were isolated from peripheral blood with a Pancoll density gradient. The percentages of freshly isolated neutrophils undergoing apoptosis or necrosis were determined by flow cytometry using Annexin V - FITC and Propidium Iodide (PI) staining. Phagocytic capacity was evaluated using pHrodo Green S. aureus BioParticles Conjugates. A Boyden's chamber model was used to determine the level of the neutrophil's mobility and chemotaxis response to 10% human serum. Formation of Neutrophils Extracellular Traps (NETs) and induction of the oxidative burst were studied upon phorbol 12-myristate 13-acetate (PMA) stimulation (25 nM) with SYTOX™ Green Nucleic Acid and flow cytometry with CellROX Green assay, respectively. The intracellular and circulating concentrations of anti-microbial compounds (Myeloperoxidase MPO, Lactotransferrin LTF, Cathelicidin-LL37, and defensins) were measured using commercially available ELISA assays. The results were evaluated with an unpaired t-test using GraphPad Prism 9. Quantitative data presented as medians (25%-75%). Results Genetic analysis identified three heterozygous variants of CLPB gene p.Arg327Trp, p.Arg628Cys, p .Arg629Cys in three different families. Two of these mutations were novel p.Arg327Trp and p .Arg629Cys. Interestingly, the p.Arg327Trp variant was located outside the ATPase domain. In total 7 family members who suffered from neutropenia and were carriers of the CLPB variants were available for neutrophil functional assays (Figure 1). Neutrophils collected from CLPB-deficient individuals have a higher rate of apoptosis as compared to healthy neutrophils: 57(52-66)% vs 23(20-27)%, P&amp;lt;0.0001, and necrosis: 0.56(0.31-1.07)% vs 0.13(0.10-0.21)%, P=0.02. CLPB-deficient neutrophils display reduced mobility in the absence of stimulation: 298(191-413) vs 1011(936-1201) migrating neutrophils, P&amp;lt;0.0001, and after 10% human serum treatment: 2090(1746-2458) vs 3736(3071-4828) migrating neutrophils, P=0.0014. Similarly, reduced phagocytosis of S. aureus bioparticles was observed in CLPB-deficient neutrophils: 1890(1679-2063) vs 3352(2472-4440) Fluorescence Units, respectively, P=0.0029. Upon PMA stimulation, neutrophils from CLPB patients produced less reactive oxygen species compared to controls: 311(175-542)FMI vs 775(766-1150)FMI, P=0.08, and their ability to produce NETs is almost entirely abolished: 0.5(0.21-0.79)ng/µl vs 1.3(1.08-3.05)ng/µl, P=0.0075. Moreover, CLPB-deficient neutrophils have reduced intracellular concentration of myeloperoxidase: 35(31-39)ng/mg vs 55(41-63)ng/mg, P=0.0043 and cathelicidin LL-37: 5.5(5-6.3)ng/µg vs 19.22(14.21-23.28)ng/µg, P=0.0003. The concertation of the LL-37 is also reduced in patients' plasma 1.34(0.84-1.78)ng/ml vs 7.17(5.38-8.03)ng/ml, P&amp;lt;0.0001. Both intracellular and circulating concentrations of LTF and defensins did not differ between study groups. Conclusion Neutrophils from neutropenic carriers of CLPB mutations are functionally impaired, especially concerning their ability for NETs formation and cathelicidin LL-37 production.

Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms.Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration.Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.