Fluorescein Research Articles

Gd-MOF composites luminescent arrays for highly sensitive detection of epileptic drug and biomarkers

Nowadays, more than 50 million people suffer from epilepsy in worldwide. Epilepsy can be diagnosed more efficiently by detecting epileptic biomarkers such as human serum albumin (HSA) and relative nucleic acid. Pregabalin is an effective curing drug for epilepsy, but the dosage of it needs to be strictly controlled. In this work, to realize highly efficiently detect and discriminate epileptic curing drugs and biomarkers (pregabalin, epilepsy-DNA-1, and HSA), a novel Gd-MOF has been synthesized by H2L ligand (H2L = 5-(4-(imidzol-1-yl)phenyl)isophthalic acid), namely [Gd2L3(DMF)(H2O)2]n, which can be further functionalized with 3-Bromo-L-phenylalanine (L-3-Br-PHE-OH), thioflavin T (ThT) and fluorescein isothiocyanate isomer I labeled anti-HSA (anti-HSA/FITC), respectively. Through the post-synthetic modification (PSM) method, three Gd-MOF composite materials with good luminescence performance can be successfully prepared, namely Gd-MOF@L-3-Br-PHE-OH (Gd-MOF-1), Gd-MOF@ThT (Gd-MOF-2), Gd-MOF@anti-HSA/FITC (Gd-MOF-3). These three composite materials can be used for the detection of pregabalin, epilepsy-DNA-1, and HSA with high selectivity and sensitivity, respectively. The limit of detection (LOD) is 45.41 nM for pregabalin, 5.56 nM for epilepsy-DNA-1, and 5.11 nM for HSA. Development of detection application of Gd-MOF composite materials for drugs and biomarkers was also performed in the real biological samples. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) results demonstrated that Gd-MOF based luminescent array can simultaneously identify and detect epileptic drugs and biomarkers in the human serum environment. Finally, detection mechanisms for this epileptic drug and biomarkers were also investigated and discussed in detail.

Interfering TRIB3 protects the blood brain barrier through PI3K/Akt pathway to alleviate cerebral ischemia-reperfusion injury in diabetes mellitus mice

This study aimed to treat diabetic cerebral ischemia-reperfusion injury (CI/RI) by affecting blood brain barrier (BBB) permeability and integrity. The CI/RI model in DM mice and a high glucose (HG) treated oxygen and glucose deprivation/reoxygenation (OGD/R) brain endothelial cell model were established for the study. Evans blue (EB) staining was used to evaluate the permeability of BBB in vivo. TTC staining was used to analyze cerebral infarction. The location and expression of tribbles homolog 3 (TRIB3) in endothelial cells were detected by immunofluorescence. Western blotting was used to detect the protein expressions of TRIB3, tight junction molecules, adhesion molecules, phosphorylated protein kinase B (p-AKT) and AKT. The levels of pro-inflammatory cytokines were detected by qRT-PCR. Trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran were used to measure vascular permeability in vitro. TRIB3 ubiquitination and acetylation levels were detected. Acetyltransferase bound to TRIB3 were identified by immunoprecipitation. TRIB3 was localized in cerebral endothelial cells and was highly expressed in diabetic CI/R mice. The BBB permeability in diabetic CI/R mice and HG-treated OGD/R cells was increased, while the junction integrity was decreased. Interference with TRIB3 in vitro reduces BBB permeability and increases junction integrity. In vivo interfering with TRIB3 reduced cerebral infarction volume, BBB permeability and inflammation levels, and upregulated p-AKT levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin reversed the effects of TRIB3-interfering plasmid. In vitro HG treatment induced TRIB3 acetylation through acetyltransferase p300, which in turn reduced ubiquitination and stabilized TRIB3. Interfering TRIB3 protects BBB by activating PI3K/AKT pathway and alleviates brain injury, which provides a new target for diabetic CI/RI.

Preparation and characterization of ball milled hexagonal‑boron nitride (BMBN) and fluorescein (FL) decorated reduced graphene oxide (RGO) nanocomposite towards toxic heavy metal ion detection

In the present work, a novel hybrid fluorescent nanocomposite (RBF) based on ball milled hexagonal-Boron Nitride (BMBN) and Fluorescein (FL) decorated reduced Graphene Oxide (RGO) was synthesized for the effective detection and removal of hazardous heavy metal ions present in effluents. Where two different textile effluents namely, reaction outlet and factory outlet collected at the river Noyyel, Tamilnadu were considered. The prepared RBF was subjected to XRD, FTIR, SEM, EDX, TEM, PSA, BET, UV–vis, PL and XPS studies which strongly confirm the decoration of FL and BMBN at the surface of RGO sheets. The selectivity of RBF was examined by considering various toxic heavy metal ions. Where the RBF exhibits remarkable sensing towards Zn2+ (10.7 nM) via enhanced fluorescence and for Fe3+ (15.6 nM), Sn4+ (60.7 nM) and Hg2+ (465.4 nM) ions by “Turn-off” fluorescence only at neutral pH. In addition, the RBF was highly efficient to remove Hg (81.3 %) than Zn (63.7 %), Fe (38.2 %) and Sn (62.9 %) in real-time samples. Moreover, the prepared RBF could also serve as water sensor for the water contaminated organic solvents of Acetonitrile (ACN) and Tetrahydrofuran (THF). Hence the prepared RBF might be a potential choice for the effective detection and removal of heavy metal ions during wastewater treatment.

Blue light impairs cornea and corneal wound healing by downregulating VCAM1 partly

This study aimed to investigate the effects of long-term pollution from different wavelengths of light on the corneal epithelium (CE) and identify potential biomarkers. Rabbits were exposed to red, green, blue, white, and environmental light for 6weeks. The CE was assessed using various techniques such as fluorescein sodium staining, transcriptome sequencing, electron microscopy, and molecular assays. In human corneal epithelial cells (hCECs), the downregulation of vascular cell adhesion molecule 1 (VCAM1) in response to blue light (BL) pollution was observed. This downregulation of VCAM1 inhibited migration, increased reactive oxygen species (ROS) levels, and apoptosis, and inhibited the AKT/p70 S6 kinase cascade in hCECs. Animal experiments confirmed that BL pollution caused similar effects on the rabbit cornea, including increased ROS production, apoptosis, delayed wound healing, and decreased VCAM1 expression. Overall, BL-induced VCAM1 downregulation may impair CE and wound healing and promote ROS and apoptosis invitro and invivo.

Assessment of Office-Based Probing with Dacryoendoscopy for Treatment of Congenital Nasolacrimal Duct Obstruction: A Retrospective Study.

We aimed to evaluate the safety and efficacy of office-based probing with dacryoendoscopy under local anesthesia for congenital nasolacrimal duct obstruction (CNLDO). This single-institution study retrospectively reviewed data on 72 eyes of 64 consecutive children (38 boys, 43 eyes; 26 girls, 29 eyes), aged between 6 and 17 (mean age: 10.0 ± 2.7) months with suspected CNLDO, from July 2016 to February 2022. These patients underwent probing with dacryoendoscopy under local anesthesia. CNLDO was diagnosed clinically based on the presence of epiphora and sticky eyes due to mucous discharge commencing within the first 3 months of life, increased tear meniscus height, and fluorescein dye disappearance test results. A total of 63 of the 72 eyes had narrowly defined CNLDO, and 9 eyes had other types of obstructions. The intervention success rate was 100% (63/63 eyes) for patients with typical CNLDO and 97.2% (70/72 eyes) for the entire study cohort. Moreover, CNLDO was classified into five types based on the features of the distal end of the nasolacrimal duct. Probing with dacryoendoscopy is safe and yields a high success rate in pediatric patients with CNLDO. This is the first study to assess the safety and efficacy of probing with dacryoendoscopy under local anesthesia in pediatric patients with CNLDO.

EXTH-25. IN VIVO TWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing B7-H3 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. B7-H3-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against B7-H3 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

Effect of fluorescein dye concentration in oral cancer tissue: Statistical and spectroscopic analysis

Oral cancer screening with exogenous agents is highly demanding due to high sensitivity, as the early diagnosis plays a vital role in achieving favorable outcomes for oral squamous cell carcinomas (OSCC) by facilitating prompt detection and comprehensive surgical removal. Optical techniques utilizing the local application of fluorescein dye or fluorescence-guided surgery offer potential for early OSCC detection. The use of fluorescein dye in oral cancer is significantly less, and there is a need to inspect the local application of fluorescein dye in oral cancer patients. Concentration-based investigations of the dye with OSCC patients are essential to ensure accurate fluorescence-guided surgery and screening with fluorescein labeling and to mitigate possible adverse effects. Additionally, analyzing the dye distribution within OSCC tissues can provide insights into their heterogeneity, a critical indicator of malignancy. The present study includes a concentration-based statistical and spectroscopic analysis of fluorescein dye in ex-vivo and in-vivo OSCC patients. In the ex-vivo examination of OSCC tissues, five concentrations (18.66 ± 0.06, 9.51 ± 0.02, 6.38 ± 0.01, 4.80 ± 0.004, and 3.85 ± 0.002 millimolar) are employed for optical analysis. The ex-vivo OSCC tissues are analyzed for multiple statistical parameters at all concentrations, and the results are thoroughly described. Additionally, spectroscopic analysis is conducted on all concentrations for a comprehensive evaluation. Following optical analysis of all five concentrations in the ex-vivo study, two concentrations, 6.38 ± 0.01 and 4.80 ± 0.004 millimolar, are identified as suitable for conducting in-vivo investigations of oral cancer. A detailed spectroscopic and statistical study of OSCC tissues in-vivo has been done using these two concentrations.

Effect of sodium fluorescein strip application on cornea parameters commonly used in laser-assisted in-situ keratomileusis

To assess the effect of sodium fluorescein (NaF) strip on corneal parameters commonly used in Laser-assisted in-situ keratomileusis (LASIK). Eighty-six subjects (172 eyes) scheduled for LASIK were recruited between January and March 2022. The study and statistical analysis test were conducted in April 2022. Topographic measurements of corneal parameters, including central corneal thickness (CCT), anterior keratometric (K) readings (K1, flat keratometry; K2, steep keratometry), horizontal corneal diameter (white to white, WTW), and corneal asphericity (Q value), were obtained using a Scheimpflug device (Pentacam) before and 10 min after NaF strip treatmentThe Pentacam recorded a small significant increase in CCT (mean 538.88 ± 28.78 μm to 547.90 ± 29.94 μm; p < .001), with no differences in K1 and K2 (mean 42.24 ± 1.35D to 42.24 ± 1.35D, and mean 43.34 ± 1.50D to 43.32 ± 1.51D; P > .05, for all) as well as WTW(mean 11.58 ± 0.32 mm to 11.58 ± 0.32 mm, P > .05) before and after NaF strip intervention. Furthermore, there was no significant difference was observed in Q value (mean − 0.30 ± 0.13 to − 0.30 ± 0.14, P > .05). These results indicate that clinicians should avoid NaF strip application before obtaining precise topographic measurements of cornea parameters using the Pentacam.

Neurovascular unit disruption and blood–brain barrier leakage in MCT8 deficiency

BackgroundThe monocarboxylate transporter 8 (MCT8) plays a vital role in maintaining brain thyroid hormone homeostasis. This transmembrane transporter is expressed at the brain barriers, as the blood–brain barrier (BBB), and in neural cells, being the sole known thyroid hormone-specific transporter to date. Inactivating mutations in the MCT8 gene (SLC16A2) cause the Allan-Herndon-Dudley Syndrome (AHDS) or MCT8 deficiency, a rare X-linked disease characterized by delayed neurodevelopment and severe psychomotor disorders. The underlying pathophysiological mechanisms of AHDS remain unclear, and no effective treatments are available for the neurological symptoms of the disease.MethodsNeurovascular unit ultrastructure was studied by means of transmission electron microscopy. BBB permeability and integrity were evaluated by immunohistochemistry, non-permeable dye infiltration assays and histological staining techniques. Brain blood-vessel density was evaluated by immunofluorescence and magnetic resonance angiography. Finally, angiogenic-related factors expression was evaluated by qRT-PCR. The studies were carried out both in an MCT8 deficient subject and Mct8/Dio2KO mice, an AHDS murine model, and their respective controls.ResultsUltrastructural analysis of the BBB of Mct8/Dio2KO mice revealed significant alterations in neurovascular unit integrity and increased transcytotic flux. We also found functional alterations in the BBB permeability, as shown by an increased presence of peripheral IgG, Sodium Fluorescein and Evans Blue, along with increased brain microhemorrhages. We also observed alterations in the angiogenic process, with reduced blood vessel density in adult mice brain and altered expression of angiogenesis-related factors during brain development. Similarly, AHDS human brain samples showed increased BBB permeability to IgG and decreased blood vessel density.ConclusionsThese findings identify for the first time neurovascular alterations in the MCT8-deficient brain, including a disruption of the integrity of the BBB and alterations in the neurovascular unit ultrastructure as a new pathophysiological mechanism for AHDS. These results open a new field for potential therapeutic targets for the neurological symptoms of these patients and unveils magnetic resonance angiography as a new non-invasive in vivo technique for evaluating the progression of the disease.

Microalgae polysaccharides exert antioxidant and anti-inflammatory protective effects on human intestinal epithelial cells in vitro and dextran sodium sulfate-induced mouse colitis in vivo

Microalgae polysaccharides (MAPS) have emerged as novel prebiotics, but their direct effects on intestinal epithelial barrier are largely unknown. Here, MAPS isolated from Chlorella pyrenoidosa, Spirulina platensis, and Synechococcus sp. PCC 7002 were characterized as mainly branched heteropolysaccharides, and were bioavailable to Caco-2 cells based on fluorescein isothiocyanate labeling and flow cytometry analysis. These MAPS were equally effective to scavenge hydroxyl and superoxide radicals in vitro and to attenuate the H2O2-, dextran sodium sulfate-, tumor necrosis factor α-, and interleukin 1β-induced burst of intracellular reactive oxygen species and mitochondrial superoxide radicals, interleukin-8 production, cyclooxygenase-2 and inducible nitric oxide synthase expression, and/or tight junction disruption in polarized Caco-2 cells. MAPS and a positive drug Mesalazine were intragastrically administered to C57BL/6 mice daily for 7 d during and after 4-d dextran sodium sulfate exposure. Clinical signs and colon histopathology revealed equivalent anti-colitis efficacies of MAPS and Mesalazine, and based on biochemical analysis of colonic tight junction proteins, goblet cells, mucin 2 and trefoil factor 3 transcription, and colonic and peripheral pro-inflammatory cytokines, MAPS alleviated dextran sodium sulfate-induced intestinal epithelial barrier dysfunction, and their activities were even superior than Mesalazine. Overall, MAPS confer direct antioxidant and anti-inflammatory protection to intestinal epithelial barrier function.

Factor VIII Is an Endothelial Factor That Promotes Vessel Stability

Background: Hemophilia A (HA) is a rare bleeding disorder caused by the absence or dysfunction of factor VIII (FVIII). Clinical manifestations include spontaneous bleedings that primarily consist of hemarthroses and intracranial hemorrhages. Attenuated microvascular endothelial functionality is observed in HA patients suggesting a dysfunction in endothelial cells (ECs). Current therapeutic practice consists of replacement therapy with recombinant FVIII (rFVIII), either as needed to treat acute hemorrhages, or as prophylaxis to prevent bleeds. It is well established that FVIII is largely secreted by ECs and in particular by sinusoidal endothelial cells in the liver. Healthy and HA ECs can be isolated from the peripheral blood as blood outgrowth endothelial cells (BOECs) or can be differentiated from induced pluripotent stem cells (iPSCs). Here we show that, in addition to its role in blood coagulation, FVIII plays a role in endothelial cell functionality. Aims: To elucidate the potential impact of FVIII on EC stability and to investigate the effect of different rFVIII products on the maintenance of EC functionality. Methods: BOECs isolated from HA patients and healthy donors were used. HA BOECs were treated in vitro with B-domain deleted (BDD; simoctocog alfa) or full-length (FL) rFVIII products, and EC functionality was evaluated by tubulogenic, migration, permeability and proliferation assays. The impact of the different rFVIII products on vessel formation and permeability was evaluated in vivo in NOD/SCID γ-Null (NSG) and NOD/SCID γ-Null HA (NSG-HA) mice. Results: In vitro results demonstrated a weakening in tubulogenesis, migration potential and permeability in HA ECs vs healthy ECs and a significant enhancing of tubule network formation in HA BOECs treated with BDD-rFVIII or FL-rFVIII. Quantification of nodes, junctions, branches and the total length of the vessel-like structures confirmed that treatment of HA BOECs with BDD-rFVIII or FL-rFVIII improved HA BOEC function. Moreover, migration assays of HA BOECs treated with BDD-rFVIII or FL-rFVIII showed that the migration ability of HA BOECs was improved after treatment with either of the rFVIII products. We also evaluated whether rFVIII could ameliorate cell permeability. When treated with either rFVIII, the HA BOEC monolayer was less permeable to fluorescein isothiocyanate (FITC)-labeled dextran compared with that from non-treated HA BOECs. Vessel permeability assays in vivo demonstrated a significant reduction of dye extravasation in NSG-HA mice treated with rFVIII, with an improvement in mice treated with BDD-rFVIII compared with FL-rFVIII. Conclusion: In conclusion, information about the possible extra-coagulative role of FVIII may be crucial to understand the key molecular targets missing in HA patients at the cellular level that impair EC functionality. Knowledge of the possible effect of different rFVIII products on ECs functionality can lead to new therapeutic approaches potentially resulting in safer and more efficient treatment of HA.

What have we learned in fluorescein-guided resection of brain metastases? An update after 79 consecutive cases.

Cerebral metastasis (CM) is the most common malignancy affecting the brain. Individualized treatment of CM still represents a challenge for neuro-oncological teams: in patient eligible for surgery, complete tumor removal is the most relevant predictor of overall survival (OS) and neurological outcome. The development of surgical microscopes harboring specific filter able to elicit the fluorescent response from sodium fluorescein (SF) has facilitated fluorescein-guided microsurgery and the identification of pathological tumor tissue, especially at the tumor margins. In this study, we analyzed the effect of SF on the visualization and resection of a large monoinstitutional cohort of CM. Surgical database of FLUOCERTUM study (Besta Institute, Milan, Italy) was retrospectively reviewed to find CM surgically removed with a fluorescein-guided technique from March 2016 to December 2022. SF was intravenously injected (5 mg/kg) immediately after induction of general anesthesia. Tumors were removed using a microsurgical technique with the YELLOW560 filter (Carl Zeiss Meditec, Oberkochen, Germany). In the most recent cases, biopsies at the tumor margins were performed to evaluate the ability of fluorescein to discriminate between fluorescent and nonfluorescent tissue at the lesion borders. Seventy-nine patients were included; most of them showed a bright, diffuse fluorescent staining that markedly enhanced tumor visibility; 11 melanomas presented a specific faint enhancement of the black pigmented central nodule with high fluorescence at tumor boundaries. Only in a minimal percentage of cases (N.=4-5.1%), fluorescein enhancement was tenuous, thus not providing a significant help during tumor resection. Altogether, in more than 90% of cases, SF was considered useful in the identification of tumoral tissue and in achieving a high rate of CM resection; thus, gross total resection was achieved in 96.2% (N.=76) of patients and in no case the detection of tumor remnants was an unexpected event. The resulted sensitivity and specificity of fluorescein in identifying tumor tissue at the tumor margin was 88.9% with a predictive positive value of 88.9%. No adverse event was registered during the postoperative course. The use of SF is a valuable method for safe fluorescence-guided tumor resection. Our data showed a positive effect of fluorescein-guided surgery on intraoperative visualization during resection of CM, suggesting a role in improving the extent of resection of these lesions.

Development of a recombinase-aided amplification method combined with lateral flow dipstick assay to detect Porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is a pathogenic virus that suppresses the immune system of pigs, impacting their health and causing economic losses. Rapid diagnostic tools for early detection of PCV2 are critical to disease prevention and control. Several molecular techniques have been established for detecting PCV2 but costly equipment and time-consuming methods are unsuitable for field inspection. In this study, we developed a recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay to compare with polymerase chain reaction (PCR) and quantitative PCR (qPCR) in detecting PCV2 in suspected field samples. To amplify RAA products, 15 primer pairs were designed from the conserved region of the open reading frame (ORF) 1 gene based on multiple alignments of eight PCV2 genotypes. The most efficient primer pair and conditions for the RAA-LFD assay were tested and selected. Limit of detection, repeatability, and reproducibility were determined using the constructed plasmid. DNA was extracted from positive samples for specificity testing as well as from 100 field samples to compare the detection of the RAA-LFD assay with PCR and qPCR. The F1/R1 primer pair was chosen and labeled with fluorescein isothiocyanate at the 5' end of the forward primer and with biotin at the 5' end of the reverse primer. The limit of detection of the RAA-LFD assay was 10 copies/μL at 38°C for 30 min. The RAA-LFD assay was repeatable and reproducible, with no cross-reaction with PCV3, Actinobacillus pleuropneumoniae, Porcine epidemic diarrhea virus, Classical swine fever virus, Porcine reproductive and respiratory syndrome virus - North America strain (PRRSV-US) and Porcine reproductive and respiratory syndrome virus - European strain (PRRSV-EU). Based on testing with 100 samples, the developed RAA showed 100% specificity and 90.56% and 85.71% sensitivity when compared to PCR and qPCR, respectively Cohen's kappa coefficients showed a good agreement with the established techniques. The RAA-LFD assay targeting the ORF1 gene was highly sensitive, specific, quick, and simple to perform in the field.

CORM‑2 reduces cisplatin accumulation in the mouse inner ear and protects against cisplatin-induced ototoxicity

IntroductionCisplatin is a life-saving anticancer compound used to treat multiple solid malignant tumors, while it causes permanent hearing loss. There is no known cure, and the FDA has not approved any preventative treatment for cisplatin-based ototoxicity. ObjectivesThis study investigated whether the carbon monoxide (CO)-releasing tricarbonyldichlororuthenium (II) dimer, CORM-2, reverses cisplatin-induced hearing impairment and reduces cisplatin accumulation in the mouse inner ear. MethodsMale 6-week-old BALB/c mice were randomly assigned to one of the following groups: control (saline-treated, i.p.), CORM-2 only (30 mg/kg, i.p., four doses), cisplatin only (20 mg/kg, i.p., one dose), and CORM-2 + cisplatin, to determine whether cisplatin-based hearing impairment was alleviated by CORM-2 treatment. ResultsOur results revealed CORM-2 significantly attenuated cisplatin-induced hearing loss in young adult mice. CORM-2 co-treatment significantly decreased platinum accumulation in the inner ear and activated the plasma membrane repair system of the stria vascularis. Moreover, CORM-2 co-treatment significantly decreased cisplatin-induced inflammation, apoptosis, and cochlear necroptosis. Because the stria vascularis is the likely cochlear entry point of cisplatin, we next focused on the microvasculature. Cisplatin induced increased extravasation of a chromatic tracer (fluorescein isothiocyanate [FITC]-dextran, MW 75 kDa) around the cochlear microvessels at 4 days post-treatment; this extravasation was completely inhibited by CORM-2 co-therapy. CORM-2 co-treatment effectively maintained the integrity of stria vascularis components including endothelial cells, pericytes, and perivascular-resident macrophage-type melanocytes. ConclusionCORM-2 co-therapy substantially protects against cisplatin-induced ototoxicity by reducing platinum accumulation and toxic cellular stress responses. These data indicate that CORM-2 co-treatment may be translated into clinical strategy to reduce cisplatin-induced hearing loss.

Inhibition of non-specific protein adsorption on PMMA surface: The role of surface modification

Mitigating non-specific adsorption (NSA) occurring on the surface of microfluidic devices plays a critical role in the detection of early protein biomarkers for clinical diagnosis. Surface-modified methods commonly used in the formation of microfluidic channels include BSA coating, PEG grafting, and Plasma cleaning. The surface composition and microstructure of PMMA surfaces were characterized by Fourier transfer infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), water contact angles (CA), and atomic force microscopy (AFM) measurements. The NSA on surfaces of bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (FITC) were investigated by microfluidics and fluorescence microscopy. The results showed that the hydrophobicity of the PMMA surface was enhanced after modification with PEG. We further evaluated their anti-protein adsorption properties under a mild alkaline (PH 7.4) condition, including concentration, reaction time, and sample volume. The BSA coating had the best anti-protein adsorption effect, reaching over 87.6%. The anti-protein adsorption effect of the plasma cleaning method is slightly inferior to that of BSA coating, at 86.1%, but the modification process is simpler and faster. Due to the insufficient stability of PEG, its resistance to protein adsorption decreases as the concentration increases.

Microfluidic synthesis of pH-responsive molecularly imprinted silica nanospheres for fluorescence sensing target glycoprotein.

Here, fluorescent artificial antibodies for sensing ovalbumin in food were synthesized by molecular imprinting technique in a microfluidic reactor. A phenylboronic acid-functionalized silane was employed as the functional monomer to enable the polymer has pH-responsive property. Fluorescent molecularly imprinted polymers (FMIPs) could be produced continuously in a short time. Both fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RB)-based FMIPs can specifically recognize the target ovalbumin, particularly FITC-based FMIP, giving an imprinting factor of 2.5 and cross-reactivity factors of 2.7 (ovotransferrin), 2.8 (β-lactoglobulin) and 3.4 (bovine serum albumin), and was applied for the detection of ovalbumin in milk powder with recovery rates of 93-110%; moreover, the FMIP can be reused at least four times. Such FMIPs have promising future in replacing the fluorophore-labelled antibodies to fabricate fluorescent sensing devices or establish immunoassay methods, which have extra merits of low-cost, high stability and recyclability, easy to carry and store at ambient environments.

A hybridized mechano-electroporation technique for efficient immune cell engineering

Immune cell engineering, which involves genetic modification of T cells, natural killer cells, and macrophages, is shifting the paradigm in immunotherapy for treating hematologic malignancies. These modified cells can be viewed as living drugs and offer advantages, including dynamic functionality, active local trafficking, and boosting the immune system while recognizing and eliminating malignant cells. Among the current technologies employed for the modification of immune cell functions, electroporation stands as a predominant approach, but it suffers from heterogeneity arising from the treatment of a bulk population of immune cells during the manufacturing procedures. To address this challenge of the field, here we present a hybrid approach to induce consecutive gentle mechanical and electric shocks. This approach enhances the treatment homogeneity and improves outcomes in difficult-to-load immune cells. The hybrid approach aims to enhance the treatment homogeneity by passing individual immune cells through a microengineered filter membrane with micropores smaller than the cell diameter. This facilitates the creation of transient pores in the cell membrane, followed by efficient delivery of biomolecules through the complementary use of a gentle electric shock. Using this hybrid mechano-electroporation (HMEP) system, we could successfully deliver fluorescein isothiocyanate (FITC) dextran molecules from the smallest (4 kDa) to the largest (2000 kDa) size and EGFP expressing plasmid DNA into different immune cell types. We also provide insight into the delivery performance of the HMEP system in comparison with the benchtop electroporation since both methods hinge on membrane disruption as their permeabilization mechanism. Immune cells treated with the HMEP protocol demonstrated higher delivery efficiencies while maintaining cell viability compared to those experiencing conventional electroporation. Therefore, membrane-based mechanoporation can be a cost-effective and efficient approach to pre-treat the hard-to-deliver immune cells before electroporation, elevating the treatment homogeneity and delivery of exogenous cargoes to a higher level.

Assessing indoor PM2.5 microbial activity in a university campus environments in Beijing

Most people spend 90 % of their time inside, indoor bioaerosols significantly impact human health. This study aimed to investigate the substantial impact of indoor bioaerosols, particularly the harmful small particle-size components within Particulate Matter 2.5 (PM2.5), on human health in a crowded campus setting. Conducted during the summer at a Beijing university, the research assesses microbial activity within PM2.5 in offices (occupied by both teachers and students) and student dormitories, encompassing bedrooms and common areas. The optimized Fluorescein Diacetate (FDA) hydrolysis method was employed for assessment. Indoor microbial activity ranged from 1.18 to 176.56 ng m−3 sodium fluorescein while outdoor activity ranged from 6.14 to 90.15 ng m−3 sodium fluorescein. Meteorological factors, including heavy rain and strong winds, influenced the correlation between indoor and outdoor microbial activity, contrasting with the effect of air pollution levels. Offices with high occupancy and outdoor environments with strong winds or heavy rain often resulted in an I/O ratio >2, and a negative correlation was observed between the I/O ratio and indoor microbial activity. The study underscores the significance of ventilation type (air conditioning vs. natural ventilation), emphasising the importance of maintenance and clean living habits in reducing indoor microbial loads. Furthermore, it identifies population density as a pivotal factor affecting microbial activity in office air. This study contributes to assessing Indoor Air Quality (IAQ) and controlling bioaerosol pollution, and provides an effective measure for rapidly evaluating biological air pollution in crowded indoor environments using the PM-based microbial activity.

Impact of a direct-fed microbial supplementation on intestinal permeability and immune response in broiler chickens during a coccidia challenge.

Maintaining intestinal health supports optimal gut function and influences overall performance of broilers. Microlife® Prime (MLP) contains a unique combination of four strains of Bacillus spp. selected to support a healthy gut which may improve performance. The aim of this study was to determine the effects of MLP supplementation on intestinal health and immunity of broilers challenged with a mixed coccidia infection during peak [0 to 6-day post-infection (dpi)] and recovery phases (6 to 13 dpi). A total of 120 male, 4 days-old Ross 708, broiler chicks were allotted to 3 treatment groups (8 replicate cages; 5 birds/cage) in a randomized complete block design. Treatments included a non-challenge (NEG), a coccidia challenge (POS), and coccidia challenge fed MLP (5 × 105 CFU/g of diet). Diets were corn-soybean meal-based. At 11 days of age, all birds, except for NEG, were orally gavaged with 15 doses (3 × the recommended commercial dose). On 6, 9, and 13 dpi, birds were orally gavaged with fluorescein isothiocyanate conjugate dextran (FITC-d). Plasma and mid-jejunum tissues were collected 2 h later. On 6 dpi, duodenal lesions from 2 birds/cage were scored and droppings were collected for oocyst enumeration. Body weight gain (BWG) and feed conversion ratio (FCR) were calculated over the experimental period. Data were analyzed with GLIMMIX procedure of SAS. During the peak phase, POS birds had reduced BWG (23%) and FCR (15%) compared to NEG birds (P < 0.05), while birds fed MLP had similar BWG (209 and 208 g) and FCR (1.17 and 1.21) compared to NEG (P > 0.05). On 6 dpi, POS birds had higher lesion scores and oocyst shedding, 2 × increase in serum FITC-d, and higher jejunum IL-10, and IFN-γ mRNA compared to NEG (P < 0.05). Birds fed MLP had reduced plasma FITC-d compared to POS birds (P < 0.05) and similar IL-10 and IFN-γ mRNA. On 13 dpi, birds fed MLP had lower plasma FITC-d, jejunum IL-10 and IFN-γ mRNA compared to POS birds (P < 0.05), but similar IL-10 to NEG birds (P > 0.05). This study confirms MLP improves intestinal health and positively modulates mucosal immune response post-coccidia challenge.

Nanoscatterer-Assisted Fluorescence Amplification Technique.

Weak fluorescence signals, which are important in research and applications, are often masked by the background. Different amplification techniques are actively investigated. Here, a broadband, geometry-independent and flexible feedback scheme based on the random scattering of dielectric nanoparticles allows the amplification of a fluorescence signal by partial trapping of the radiation within the sample volume. Amplification of up to a factor of 40 is experimentally demonstrated in ultrapure water with dispersed TiO2 nanoparticles (30 to 50 nm in diameter) and fluorescein dye at 200 μmol concentration (pumped with 5 ns long, 3 mJ laser pulses at 490 nm). The measurements show a measurable reduction in linewidth at the emission peak, indicating that feedback-induced stimulated emission contributes to the large gain observed.