Labeling Kit Research Articles

Epigenetic mechanism of EZH2-mediated histone methylation modification in regulating ferroptosis of alveolar epithelial cells in sepsis-induced acute lung injury.

Sepsis-induced acute lung injury (SI-ALI) leads to significant deaths in critically ill patients worldwide. This study explores the mechanism of EZH2 regulating ferroptosis of alveolar epithelial cells (AECs) in SI-ALI. In vitro cell model and in vivo mouse lung injury model of sepsis were established. EZH2 expression in lung tissues was intervened by sh-EZH2, followed by H&E staining observation of lung tissue pathological changes. EZH2, H3K27me3, USP10, GPX4, and ACSL4 expressions were determined by qRT-PCR or Western blot. ROS, GSH, and iron ion levels were detected using fluorescent labeling and reagent kits, respectively. ChIP analyzed the enrichment of EZH2 and H3K27me3 on USP10 promoter. The binding between USP10 and GPX4, and the ubiquitination level of GPX4 were detected using Co-IP. EZH2 was highly expressed in lung tissues of SI-ALI mice. EZH2 silencing alleviated ALI and ferroptosis of AECs; EZH2 increased the H3K27me3 level on USP10 promoter through histone methylation. USP10 stabilized GPX4 protein expression through ubiquitination; inhibition of USP10 partially reversed the inhibitory effect of EZH2 silencing on ferroptosis of AECs. In conclusion, EZH2 depresses USP10 expression by promoting histone H3K27me3 modification on USP10 promoter, thereby enhancing ubiquitination degradation of GPX4 and ultimately facilitating ferroptosis of AECs in sepsis.

Correlation between Matrix Metalloproteinase Presence and Caries Surface Appearance.

Cariogenic bacterial acids dissolve the inorganic elements in dentine, leaving the dentine matrix exposed. Host-derived matrix metalloproteinases (MMPs) play an essential role in caries progression as they are significant regulators of extracellular matrix turnover and can degrade exposed collagen. This paper investigates the expression of MMP2 and MMP9 across various stages of caries in primary human teeth and relate this with a diagnosis recorded by the International Caries Detection and Assessment System (ICDAS). Twenty-four sections (150 μm in thickness) from extracted teeth, clinically diagnosed using ICDAS, were immunohistochemically treated with monoclonal anti-MMP2 and anti-MMP9 antibodies. Positive staining was visualised by immunofluorescence using a VectorFluor Duet Double Labeling Kit. Images from triplicate samples for each ICDAS score were analysed using ImageJ software. Collagen degradation in caries lesions was detected using a hydroxyproline assay. MMPs were weakly detected in caries with ICDAS 1-2 scores, and an insignificant increase was detected in ICDAS 3. However, a significant increase in MMP expression was seen in caries with an ICDAS score of 4-6. There was a strong positive correlation between the ICDAS score and MMP2 (r [6] = 0.86, p = 0.002) and between ICDAS and MMP9 (r [6] = 0.82, p = 0.004). Data were analysed using two-way ANOVA followed by Tukey multiple comparison test (*p < 0.05). The use of ICDAS to assess the severity of caries lesions and how this correlates with the presence of MMP in these lesions validates the modern approach to caries management with a minimally invasive concept.

Targeted recovery of male cells in a male and female same-cell mixture.

DNA mixture deconvolution in the forensic DNA community has been addressed in a variety of ways. "Front-end" methods that separate the cellular components of mixtures can provide a significant benefit over computational methods as there is no need to rely on models with inherent uncertainty to generate conclusions. Historically, cell separation methods have been investigated but have been largely ineffective due to high cost, unreliability, and the lack of proper instrumentation. However, the last decade has given rise to more innovative technology that can target and recover cells more effectively. This study focuses on the development and optimization of a method to selectively label and recover male cells in a mixture of male and female epithelial cells using a Y-chromosome labeling kit with DEPArray™ technology, whereby male cells are labeled and recovered into a single extraction-ready tube. Labeling efficiency was tested using freshly collected and aged buccal swabs where 70%-75% and 38% of male cells were labeled, respectively, with less than 1% false positives. DEPArray™ detection was assessed using single buccal epithelial cells where approximately 80% of labeled cells were identified as male. Mixtures (1:1, 1:10, male to female) yielded profiles that were predominantly single source male or those in which the male component was more easily interpreted. The male-specific labeling method was demonstrated to be both robust and reliable when used on freshly collected cells. While the DEPArray™ meditated detection and recovery had notable limitations, it still improved the interpretation of the male component in same-cell mixtures in more recently collected samples.

Discovering the Radiation Biomarkers in the Plasma of Total-Body Irradiated Leukemia Patients.

The increased risk of acute large-scale radiological exposure for the world's population underlines the need for optimal radiation biomarkers. Ionizing radiation triggers a complex response by the genome, proteome, and metabolome, all of which have been reported as suitable indicators of radiation-induced damage invivo. This study analyzed peripheral blood samples from total-body irradiation (TBI) leukemia patients through mass spectrometry (MS) to identify and quantify differentially regulated proteins in plasma before and after irradiation. In brief, samples were taken from 16 leukemic patients prior to and 24 h after TBI (2 × 2.0 Gy), processed with Tandem Mass Tag isobaric labelling kit (TMTpro-16-plex), and analyzed by MS. In parallel, label-free relative quantification was performed with a RP-nanoLC-ESI-MS/MS system in a Q-Exactive mass spectrometer. Protein identification was done in Proteome Discoverer v.2.2 platform (Thermo). Data is available via ProteomeXchange with identifier PXD043516. Using two different methods, we acquired two datasets of up-regulated (ratio ≥ 1.2) or down-regulated (ratio ≤ 0.83) plasmatic proteins 24 h after irradiation, identifying 356 and 346 proteins in the TMT-16plex and 285 and 308 label-free analyses, respectively (P ≤ 0.05). Combining the two datasets yielded 15 candidates with significant relation to gamma-radiation exposure. The majority of these proteins were associated with the inflammatory response and lipid metabolism. Subsequently, from these, five proteins showed the strongest potential as radiation biomarkers in humans (C-reactive protein, Alpha amylase 1A, Mannose-binding protein C, Phospholipid transfer protein, and Complement C5). These candidate biomarkers might have implications for practical biological dosimetry.

Organ-on-a-Chip Microfluidic Systems for Tracking Exosomal Dynamic Communication

Organ-on-a-chip microfluid systems (OCMS) are miniaturized three-dimension models of human tissue and organ, designed to recapitulate the crucial physiological and biological parameters of their corresponding in vivo parts. They have emerged as a powerful multifunctional tool for various applications such as personalized medicine, drug screening, due to its ability to show biomimetic composition, designs, and functions. Recently, OCMS have been employed to model and decode inter-organ communication via exosomes. Exosomes are biological nanovesicles with approximately 30-200 nm diameter, released from most of the cell types and participate in various cellular functions via intracellular communication and by carrying different cargoes including protein, and nucleic acids. Under pathological conditions such as cancer, the release of exosomes enhances tremendously, which are either fused or internalized by the recipient cells to elicit specific biological responses. The research pertaining to the exosomal communication has employed different methods for characterizing their release by the donor cells and uptake by the recipient cells, such as nano tracking analyzer, protein quantification, transmission electron microscopy (TEM), scanning EM (SEM), and immunogold-EM, exosome labeling kits, microbead-based flow cytometry. However, the research associated with the regulation of exosomal release and uptake has been impeded by the dearth of advanced techniques for capturing dynamics of exosomes. Here in, we discuss the advances in biosensing for tracking exosomal dynamic communication in OCMS, which will open new avenues of exosomal research using microfluidic engineering for modeling intracellular communication in OCMS.

Ponicidin Induces Apoptosis of Murine Melanoma by Inhibiting the NF-κB Signaling Pathway.

Ponicidin (PON), a diterpenoid extracted from the Chinese herb Rubescens, has been reported to be a therapeutic cytotoxic drug for the treatment of various types of human cancers. According to the statistics, the incidence of malignant melanoma is increasing year by year and the degree of malignancy is extremely high, so early treatment is very important. In the present study, we demonstrated the antitumor effect of PON on melanoma in vitro and in vivo. Cell Counting Kit-8 (CCK-8) assay was used to detect cell proliferation rate, crystal violet staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) kit was used to detect cell apoptosis, and Western blotting was used to detect the expression of apoptotic indicators and related signaling pathway proteins. Finally, the tumor-bearing mouse model was constructed. Treating melanoma B16F0 and B16F10 cells with different concentrations (10 and 20 µmol/L) of PON magnificantly decreased cell viability. In addition, PON significantly activates the expression of pro-apoptotic proteins, including cleaved-poly(ADP-ribose)polymerase (PARP) (cl.PARP), Bak and Bim proteins, and also inhibits the expression of anti-apoptotic protein Mcl-1 and nuclear transcription factor nuclear factor-kappaB (NF-κB) in melanoma cells. Lastly, PON effectively inhibits the growth of mouse xenografts in vivo. These results suggest that PON induces apoptosis of melanoma cells may be achieved by inhibiting NF-κB signal pathway, but the specific mechanism remains to be further elucidated. Taken together, PON may serve as an effective potential drug for the treatment of melanoma.

Extracellular vesicles from equine follicular fluid support cumulus expansion and alter cumulus cells’ transcriptome and viability

Expansion of the cumulus cells (CC) surrounding the oocyte is crucial for growth and maturation of a healthy oocyte, that can be fertilized and develop into an embryo. Factors that are released by CC into the follicular fluid (FF) induce paracrine signaling within the follicular environment, which is essential for proper folliculogenesis. FF is a rich source of proteins, nucleic acids or extracellular vesicles (EVs) which are considered as mediators of intercellular communication within the follicle. EVs influence target cells by interacting with membrane receptors or by internalizing and delivering molecules to cytoplasm or endosomes. Recent studies in cattle have shown an involvement of EVs in controlling cumulus expansion (Hung et al. Biol Reprod. 2015; 93(5):1-9), but this effect has to date not been observed in horses. The aim of this study was, therefore, to evaluate the effect of FF-derived EVs (ffEVs) on equine CC expansion, viability and transcriptome modulations. The material consisted of 43 slaughtered mares’ ovaries, from which 304 cumulus-oocyte complexes (COCs) were obtained. EVs were isolated from small follicles (<20 mm) and characterized as described before (Gabryś et al. Theriogenology 2022; 188:116-124). COCs were matured in vitro (IVM) in the presence (200 µg protein/ml) or absence (control) of ffEVs. For the confirmation of ffEVs internalization, particles were labeled using ExoGlow-protein EV Labeling Kit, added to maturing COCs, which were afterwards imagedusing confocal microscopy. CC expansion was assessed by measuring the COCs diameter before and after IVM and viability test was performed after IVM using staining with ethidium bromide and fluorescein diacetate. Additionally, CC were isolated after 12 hrs of IVM, after which RNA was extracted and cDNA libraries were generated with QuantSeq 3’ mRNA-Seq Library Prep Kit. Transcriptome alterations of CC were investigated with next generation sequencing and bioinformatic analysis. Confocal microscopy images show the internalization of ffEVs by both cumulus cells and oocytes. Supplementation with ffEVs had a significant impact on cumulus expansion in both compacted (Cp, p<0.0001) and expanded (Ex, p<0.05) COCs. There were no differences in diameters of COCs from the control groups measured before and after IVM. The results of viability test indicate a divergent effect of COCs supplementation, as in Cp group ffEVs highly significantly increased (p<0.01), while in Ex decreased (p<0.05) CC viability compared to the control group. Despite the observed differences in CC physiology, transcriptome analysis demonstrate that CC RNA profile is slightly affected in the supplemented group. Nevertheless, the differently expressed genes comprise ones with processes and functions (e.g. cellular and metabolic processes), which may be important for the properties of cumulus and hence the maturation of the oocyte.

Extended DNA Fibers for High-Resolution Mapping.

DNA fiber-FISH is an easy and simple light microscopic method to map unique and repeat sequences relative to each other at the molecular scale. A standard fluorescence microscope and a DNA labeling kit are sufficient to visualize DNA sequences from any tissue or organ. Despite the enormous progress of high-throughput sequencing technologies, DNA fiber-FISH remains a unique and indispensable tool to detect chromosomal rearrangements and to demonstrate differences between related species at high resolution. We discuss standard and alternative steps to easily prepare extended DNA fibers for high-resolution FISH mapping.

Role of brain and muscle ARNT-like protein 1 in the rat periodontitis-induced liver injury model

Objective: Brain and muscle ARNT-like protein 1 (BMAL1) is a core component of hepatocyte molecular clock and plays an important role in the regulation of other related rhythmic genes in the body through a transcriptional-translational feedback loop in molecular circadian oscillations. Therefore, the aim of this study was to investigate the role of BMAL1 in the rat periodontitis-induced liver injury. Methods: Twelve male Wistar rats were divided into the control group and the periodontitis group according to the random number table method. The rats in the control group were untreated. The periodontitis models were established by ligating the necks of the bilateral maxillary first molars in the periodontitis group rats. After 8 weeks, periodontal clinical indexes of rats in both groups were examined and executed. Micro-CT scans of the maxilla were performed and levels of the alveolar bone resorption were analyzed. Pathological changes in periodontal and liver tissue of rats in two groups were detected by HE and oil red O staining. Biochemical kits were used to detect glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total cholesterol (TC) and triglycerides (TG) in serum. The gene and protein expression levels of BMAL1, nuclear factor kappa-B (NF-κB) and tumor necrosis factor-α (TNF-α) in liver tissue were measured by real time fluorescent quantitative-PCR (qRT-PCR), immunohistochemistry (IHC) and Western blotting (WB) assays. Apoptosis was detected in liver tissues by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) kit staining. Results: The results of HE staining of maxillary first molars and micro-CT results of maxillary bones showed that alveolar bone resorption was significant in the periodontitis group of rats. The liver histopathology results showed infiltrated inflammatory cells in the liver tissue, disorganized liver cords and a large number of lipid droplets formed in the hepatocytes of the periodontitis group compared with the control group. The results of serum biochemical assay showed that the levels of GOT [(62.77±2.59) U/L], GPT [(47.54±1.04) U/L], TC [(3.19±0.23) mmol/L] and TG [(1.11±0.09) mmol/L] in the serum of rats with periodontitis were significantly higher than that in the control group respectively [GOT: (38.66±2.47) U/L, GPT: (31.48±1.57) U/L, TC: (1.60±0.05) mmol/L and TG: (0.61±0.09) mmol/L](P=0.003, P=0.001, P=0.002, P=0.038). qRT-PCR results showed that the mRNA expression level of BMAL1 was significantly decreased in liver tissue of the periodontitis group [(0.60±0.04)%] compared to the control group [(1.01±0.07)%] (t=4.80, P=0.009), while the mRNA expression levels of NF-κB and TNF-α [(1.62±0.12)%, (2.69±0.16)%] were significantly increased compared to the control group [(1.00±0.03)%, (1.03±0.16)%] (P=0.008, P=0.002); IHC results showed that the protein expression level of BMAL1 in liver tissue of the periodontitis group (averaged optical density, AOD) (11.58±2.15) was down-regulated compared to the control group (AOD) (22.66±1.67) (P=0.015), while NF-κB and TNF-α (AOD) (31.77±2.69, 24.31±2.32) were up-regulated compared to the control group (AOD) (19.40±1.82, 11.92±0.94) (P=0.019, P=0.008). WB results showed that the protein expression level of BMAL1 in liver tissue was down-regulated in the periodontitis group [(0.63±0.10)%] compared to the control group [(1.00±0.06)%] (t=3.19, P=0.033), while NF-κB and TNF-α [(1.61±0.12)%, (2.82±0.23)%] were up-regulated compared to the control group [(1.00±0.12)%, (1.00±0.11)%] (P=0.022, P=0.002). TUNEL staining showed increased apoptotic cells in the liver tissue of the periodontitis group of rats compared to the control group. Conclusions: Periodontitis may induce liver injury by down-regulating the BMAL1 expression levels in liver tissue, which in turn activates NF-κB signaling molecules, leading to the elevated levels of inflammation and apoptosis in rat liver.

Disulfiram inhibits liver fibrosis in rats by suppressing hepatic stellate cell activation and viability

BackgroundLiver fibrosis is a wound-healing response to chronic injury, featuring with excess accumulation of extracellular matrix secreted by the activated hepatic stellate cells (HSC). Disulfiram (DSF), also known as Antabuse, has been used for the treatment of alcohol addiction and substance abuse. Recently, overwhelming studies had revealed anti-cancer effects of DSF in multiple cancers, including liver cancer. But the actual effects of DSF on liver fibrosis and liver function remain unknown.MethodsIn this study, we evaluated the effects of low-dose DSF in CCl4- and Bile Duct Ligation (BDL)—induced hepatic fibrosis rat models. Cell proliferation was detected by using the Cell-Light™ EdU Apollo®567 Cell Tracking Kit. Cell apoptosis was analyzed using a TdT-mediated dUTP nick end labeling (TUNEL) kit, viability was measured with Cell Counting Kit-8(CCK8). Relative mRNA expression of pro-fibrogenic was assessed using quantitative RT-PCR. The degree of liver fibrosis, activated HSCs, were separately evaluated through Sirius Red-staining, immunohistochemistry and immunofluorescence. Serum alanine aminotransferase (ALT) and asparagine aminotransferase (AST) activities were detected with ALT and AST detecting kits using an automated analyzer.ResultsLiver fibrosis was distinctly attenuated while liver functions were moderately ameliorated in the DSF-treated group. Activation and proliferation of primary rat HSCs isolated from rat livers were significantly suppressed by low-dose DSF. DSF also inhibited the viability of in vitro cultured rat or human HSC cells dose-dependently but had no repressive role on human immortalized hepatocyte THLE-2 cells. Interestingly, upon DSF treatment, the viability of LX-2 cells co-cultured with THLE-2 was significantly inhibited, while that of THLE-2 co-cultured with LX-2 was increased. Further study indicated that HSCs apoptosis was increased in DSF/CCl4-treated liver samples. These data indicated that DSF has potent anti-fibrosis effects and protective effects toward hepatocytes and could possibly be repurposed as an anti-fibrosis drug in the clinic.ConclusionsDSF attenuated ECM remodeling through suppressing the transformation of quiet HSCs into proliferative, fibrogenic myofibroblasts in hepatic fibrosis rat models. DSF provides a novel approach for the treatment of liver fibrosis.

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

Plasma MicroRNAs as noninvasive diagnostic biomarkers in patients with Brugada syndrome.

BackgroundBrugada syndrome (BrS) can be diagnosed by a type 1 BrS tracing in a 12-lead electrocardiogram (ECG). However, there are daily variations in the ECGs of BrS patients, which presents a challenge when diagnosing BrS. Although many susceptibility genes have been identified, the SCN5A gene is reportedly the main causative gene of BrS. However, most patients do not have an evidence of genetic predisposition to develop BrS. In addition, the diagnosis and risk stratification for ventricular fibrillation (VF) in patients with BrS presents some problems. Meanwhile, circulating micro RNAs (miRNAs) have drawn increased attention as potential biomarkers of various diseases. We hypothesize that circulating miRNAs may be potential diagnostic biomarkers for BrS.MethodsWe enrolled 70 Japanese BrS patients and 34 controls for the screening cohort. A total of 2,555 miRNA sequences were detected using the 3D-Gene miRNAs labeling kit and 3D-Gene Human miRNAs Oligo Chip. We compared the expression of the miRNAs between the BrS patients and the controls. We validated whether the miRNA were significantly up- or downregulated in the screening cohort using RT-PCR. We also enrolled 72 Japanese BrS patients and 56 controls to replicate these miRNAs.ResultsEight miRNAs (hsa-miR-223-3p, hsa-miR-22-3p, hsa-miR-221-3p, hsa-miR-4485-5p, hsa-miR-550a-5p, hsa-miR-423-3p, hsa-miR-23a-3p, and hsa-miR-30d-5p) were downregulated, and one miRNA (hsa-miR-873-3p) was upregulated by more than 3-fold in BrS patients. The multivariate logistic regression analysis determined that hsa-miR-423-3p, hsa-miR-223-3p, and hsa-miR-23a-3p were independently associated with BrS (P < 0.0001). The AUC based on cross validation was 0.871 with a sensitivity and specificity of 83.5% and 81.1%, respectively.ConclusionsThe plasma miRNAs are potential noninvasive biomarkers of BrS, and the constructed logistic model was useful for discriminating BrS.

Depletion of mmu_circ_0001751 (circular RNA Carm1) protects against acute cerebral infarction injuries by binding with microRNA-3098-3p to regulate acyl-CoA synthetase long-chain family member 4

ABSTRACT Circular RNAs (circRNAs) play a critical role in acute cerebral infarction (ACI). Our research discussed the effect of circ-Carm1 in ACI and its potential molecular mechanisms. Healthy controls and patients with ACI were included in this study. The establishment of an oxygen and glucose deprivation/reoxygenation (OGD/R) model of HT22 cells was conducted to mimic ACI in vitro. Quantitative reverse transcription polymerase chain reaction was conducted to determine mRNA levels extracted from serum and HT22 cell samples, and Western blotting was performed to determine protein levels. Terminal deoxynucleotidyl transferase dUTP nick end labeling and cell counting kit 8 assays were conducted to evaluate cellular functions. Concentrations of Fe2+ and malondialdehyde, and levels of transferrin receptor 1, glutathione peroxidase 4, and glutathione were evaluated to determine ferroptosis in OGD/R-induced HT22 cells. The binding relationships between mRNAs and miRNAs were verified. circ-Carm1 was highly expressed in OGD/R-treated HT22 cells. Deficiency of circ-Carm1 restored cell viability and suppressed ferroptosis in OGD/R-induced HT22 cells. miR-3098-3p was predicted to be a target of circ-Carm1. The miR-3098-3p inhibitor partly neutralized the functions of circ-Carm1 in OGD/R-induced HT22 cells. Furthermore, acyl-CoA synthetase long-chain family member 4 (ACSL4) was confirmed to be a downstream target of miR-3098-3p and was elevated in OGD/R-induced HT22 cells. Overexpression of ACSL4 mitigated the functions of miR-3098-3p and accelerated HT22 cell dysfunction. Hence, circ-Carm1 is upregulated in ACI. circ-Carm1 suppression protects HT22 cells from dysfunction by inhibiting ferroptosis. Therefore, inducing circ-Carm1 deficiency may be a promising therapeutic method for ACI.

Analysis of Constituent Elements and Brownian Motion of the Conjugates of anti-CD3 Antibody and Magnetic Nanoparticles

An Fc-directed conjugate (mAb-MNP) of the anti-CD3 monoclonal antibody (mAb), Foralumab, and the surface amino groups on magnetic nanoparticles (MNPs) was prepared using the SiteClick antibody labeling kit. The conjugate was imaged by transmission electron microscopy and analyzed using energy dispersive spectrum and a nanoparticle tracking analyses. The average values of drift velocities (pix/frame) due to the Brownian motion of MNPs and mAb-MNPs in phosphate-buffered saline were 3.16/0.76 and 6.70/1.98 on the x-/yaxes, respectively. Two different hydrodynamic diameters of 180.0 nm and 258.8 nm were found for the MNPs and the mAb-MNPs with an actual size of 35 nm, respectively. The drift velocity was 2 times (2×) higher for mAb-MNPs than MNPs in proportion to the 2× higher value of the square of the hydrodynamic diameter of the conjugate relative to the MNPs as calculated by Stokes

Sodium Danshensu promotes the healing of stage 2 pressure injury wounds in ischemia/reperfusion injury rat models: possible regulation of apoptosis and inflammatory response.

To investigate the protective effect and possible mechanism of sodium Danshensu (SDSS) against pressure injury caused by ischemia/reperfusion (I/R) injury. Sprague-Dawley rats were randomly divided into five groups of eight rats each: control group, model group, 10 mg/kg SDSS-treated group, 20 mg/kg SDSS-treated group, and 40 mg/kg SDSS-treated group. We used two round ferrite magnetic plates of 15 mm diameter and 3 mm thickness to establish stage 2 pressure injury model rats. Each rat was subjected to five cycles of ischemia and reperfusion to induce pressure injury. One cycle consisted of 2 h of ischemia and 0.5 h of reperfusion, which meant that each cycle included 2 h of pressure and 0.5 h of pressure relief. The outline of the wound was delineated by butter paper and marker pen, and histopathological changes were observed by hematoxylin and eosin staining. In addition, the number of apoptotic cells and the activity of caspase-3 were assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling and caspase-3 assay kits, respectively. The expression of apoptosis-regulatory proteins and inflammatory mediators was investigated by enzyme-linked immunosorbent assay. Results showed that treatment with SDSS for 7 d after establishing the pressure injury model remarkably improved the healing rate of the wound. SDSS also inhibited the levels of tumor ne- crosis factor-α, myeloperoxidase, and intercellular cell adhesion molecule-1; decreased the number of apoptotic cells; increased the ratio of B-cell lymphoma-2 (Bcl-2) / Bcl-2-associated X (Bax); and regulated the expression and activity of caspase-3. Our results suggest that SDSS exhibits a treatment efficacy for pressure injury caused by I/R injury possibly by inhibiting apoptosis and inflammatory response.

Abstract LB069: Development of cMET/cMET/EGFR Trispecific antibody as therapeutic modality for Non-small Cell Lung Cancer

Abstract Background: Tyrosine kinase inhibitors (TKIs) have been developed against EGFR. However, in NSCLC (Non-small Cell Lung Cancer) and some other cancers, the occurrence of activating mutations in EGFR leads to acquired resistance. Anomalous cMET signaling additionally contributes to developing resistance to targeted therapies, including ones directed at EGFR. A number of bispecific antibodies targeting EGFR and cMET are in clinical trials and JNJ-61186372 (Amivantamab) from Janssen labs, has been given fast track approval by the FDA. In this study we created bispecific and trispecific antibody formats which target both cMET and EGFR. Trispecific (TsAb), unlike the bispecific (BsAb), binds to two different epitopes on cMET and one on EGFR resulting in enhanced internalization of the receptors. This study demonstrates that our antibodies exhibit enhanced capabilities than the benchmark at clinical stage in cell and non-cell based in vitro assays. Methods: The two cMET binding arms of TsAb were designed to block the cMET-HGF (ligand) interaction and enhance the internalization of the receptors. Cell based assays were performed using a NSCLC cell line NCI-H1975 (ATCC CRL-5908) and HGF-expressing NCI-H1975. Receptor binding and receptor/ligand blocking (cMET/HGF and EGFR/EGF) assays were performed using both ELISA and flow cytometry. Phosphorylation assays were performed using ELISA and Western blotting. Receptor internalization studies were executed using the pHrodo labelling kit and were further assessed through fluorescence microscopy and quantified through flow cytometry. ADCC assays (Antibody dependent cellular cytotoxicity) were performed through the Crystal violet method. Results: BsAb was more effective than benchmark in terms of its binding capabilities to cMET and EGFR receptors and also in blocking the interaction of cMET and EGFR with their ligands. Moreover, functional assays revealed that the new BsAb is in most cases better than benchmark in successfully inhibiting the phosphorylation of not only the cMET and EGFR receptors but also the downstream signaling pathways which are involved in cancer cell proliferation, survival and obstructing the apoptotic pathways. Furthermore, the new TsAb was found to be more effective than BsAb and benchmark in cMET/EGFR receptor internalization which may lead to additional extenuation of the downstream pathways for these receptors. Cancer cell killing assays for ABT BsAb show ADCC comparable to the benchmark. Conclusion: Comparing to BsAbs, TsAb induces enhanced internalization of the cMET/EGFR receptors leading to further extenuation of the downstream oncogenic signaling pathways. These data suggest the development of ABT BsAb and specifically TsAb as a new drug target for patients with lung cancer and NSCLC in particular. Citation Format: Mohd Zaman, Cai Huang, Sachith Gallolu Kankanamalage, Amit K. Chaudhary, Jianbo Dong, Yue Liu. Development of cMET/cMET/EGFR Trispecific antibody as therapeutic modality for Non-small Cell Lung Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB069.

Benefits of photopolymerizable hydrogels for the effective delivery of raav vectors in human bone marrow derived mesenchymal stem cells

Purpose: Injured adult articular cartilage has a limited ability for self-healing. Administration of genetically modified bone marrow-derived mesenchymal stromal cells (MSCs) in cartilage defects is a promising approach to enhance cartilage repair, especially when using clinically relevant recombinant adeno-associated virus (rAAV) vectors [1,2]. Yet, the presence of neutralizing antibodies against the viral capsid proteins in patients [3] may impair effective gene transfer in translational protocols. Scaffold-guided rAAV gene transfer has the potential to overcome such an issue by protecting the vectors from neutralization while increasing their gene transfer efficacy by controlling their spatial and temporal delivery [4-7]. Here, we examined the feasibility of delivering rAAV vectors in human MSCs (hMSCs) via photopolymerizable hydrogel systems [8,9] as future, effective platform for cartilage repair. Methods: rAAV vectors were packaged, purified, and titrated as previously described [10,11]. rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene and rAAV-RFP the Discosoma sp. red fluorescent protein gene (RFP) [10,11]. Cy3 labeling of the rAAV vectors was performed using a Cy3 Ab Labeling Kit (Amersham/GE Healthcare) with detection by live fluorescence imaging as previously described [12]. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty (n = 2). hMSCs were prepared as previously described [10,11]. Cells (passage 1) were seeded in 48-well plates (5,000 cells/well), maintained in DMEM, 10% FBS, 100 U/ml penicillin G, 100 μl/ml streptomycin, and incubated at 37ºC during 12 h before addition of the various hydrogel systems. The hydrogel systems (FID119 F3 and FID119 F4) were prepared by overnight dissolution of the compounds (300 mg of FID119 F3, i.e. F3; 200 mg of FID119 F4, i.e. F4) 10 ml NaCl 0.9% (w/v). The F3 and F4 hydrogel systems were mixed with rAAV-lacZ or rAAV-RFP in equal parts using a BTC® Medical Europe UV-Lamp (5 min) and directly applied to the cultures for up to 21 days. Release of rAAV from the hydrogels was evaluated by AAV Titration ELISA (Progen) in conditioned culture medium using a permeable support system as previously described [12]. Transgene (RFP) expression was detected by live fluorescence imaging [10,11]. Cell viability was monitored using the Cell Proliferation reagent WST-1 (Roche Applied Science) [12]. Each condition was performed in duplicate in two independent experiments. Results: The F3 and F4 hydrogels (Fig. 1A) were both capable of encapsulating rAAV as noted by effective detection of Cy3 vector labeling in the hydrogel systems relative to control conditions (encapsulation of unlabeled vectors) (Fig. 1B). An analysis of the rAAV release profiles (Fig. 1C) showed that both the F3 and F4 hydrogels effectively released rAAV over a prolonged period of time (21 days, the longest time-point evaluated) compared with hydrogels lacking rAAV vectors, with hydrogel-free rAAV vectors, and with a condition lacking both the hydrogel and rAAV. Specifically, the F4 hydrogel system was the most potent to support a controlled release of rAAV over time (Fig. 1C). An estimation of transgene (RFP) expression upon direct contact of the hydrogels with hMSCs over time revealed that rAAV-RFP released from the F3 and F4 hydrogels promoted an effective overexpression of the RFP reporter gene in the cells for at least 21 days (the longest time-point examined) versus the various control conditions tested (Fig. 2). Equally important, an analysis of the cell proliferation indices in the cells showed no deleterious effects of the F3 or F4 hydrogel systems carrying rAAV relative to the various control conditions tested (Fig. 3). Conclusions: Controlled delivery of rAAV via FID119 hydrogels (especially with the F4 hydrogel system) has the potential to safely and durably modify hMSCs. Combining rAAV gene transfer with the use of photopolymerizable (FID119) hydrogels may provide novel, promising tools to enhance cartilage repair by implanting MSCs modified to overexpress therapeutic (chondroregenerative) genes via controlled delivery of rAAV using in particular the F4 hydrogel. REFERENCES: [1] Cucchiarini & Madry, Biomed Mater Eng. 2010, 20:135 [2] Frisch et al., Curr Stem Cell Res Ther. 2015, 10:121 [3] Cottard et al., J Clin Immunol. 2004, 24:162 [4] Rey-Rico & Cucchiarini, Acta Biomater. 2016, 29:1 [5] Rey-Rico & Cucchiarini, Curr Gene Ther. 2017, 17:127 [6] Cucchiarini & Madry, Nat Rev Rheumatol. 2019, 15:18 [7] Madry et al., Adv Mater. 2020, 32:e1906508 [8] Rey-Rico et al., Biomed Res Int. 2016, 2016:1215263 [9] Meng et al., J Exp Orthop. 2019, 6:47 [10] Cucchiarini et al., Tissue Eng Part A. 2011, 17:1921 [11] Venkatesan et al., Int J Mol Sci. 2018, 19:2635 [12] Rey-Rico et al., ACS Appl Mater Interfaces. 2016, 8:20600. ACKNOWLEDGMENTS: Supported by a grant from German Osteoarthritis Foundation ( Deutsche Arthrose-Hilfe e.V. to MC).View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

VIABILITY AND DISTRIBUTION EVALUATION OF MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE MARKED BY NANOCRYSTALS IN HORSES WITH CHRONIC LAMINITIS 24 HOURS AFTER REGIONAL INFUSION - PRELIMINARY STUDY

<h3>Background</h3> The objective of this study was to evaluate the viability, distribution and setting of previously characterized and differentiated equine Ad-MSC labeled with the Qtracker cell labeling kit after infusion into the forelimb of horses with chronic laminitis. <h3>Methods</h3> Subcutaneous fat from healthy horse was collected, and Ad-MSC were isolated and cultured. In the third pass, Ad-MSC were marked with fluorescent nanocrystals. Labeling was carried out according to the manufacturer‘s instructions. Briefly, two million Ad-MSC were marked by adding 2ul of nanocrystals, 2ul of PBS and 200ul of DMEM medium, followed by incubation at 37°C and 5% CO₂ for 1 hour. Cell viability was determined by staining with trypan blue before staining and at 1 and 24 hours after staining. 20 million allogeneic Ad-MSC marked according to the manufacturer‘s standards and suspended with 20 ml of PBS were infused in 2 horses with chronic laminitis. The infusion was performed only in one member (treated), previously drawn, by regional limb perfusion (RLP) and the tourniquet was maintained for 30 minutes. In the contralateral limb, only PBS was infused. Lamellar tissue biopsies were performed 24 hours later. The absorption of nanocrystals in the cell was observed through fluorescence microscopy in samples of labeled cells and lamellar tissue obtained by biopsy. <h3>Results</h3> Cytoplasmic absorption of nanocrystals was observed 1 and 24 hours after staining. The viability of Ad-MSC in culture before labeling was 91%. After 1 hour of incubation with nanocrystals, viability decreased slightly to 85% followed by 83% viable cells 24 hours after incubation. The results of this study demonstrate that Qdot nanocrystals can be successfully introduced into equine Ad-MSC with minimal cytotoxic effect on these cells. The cytoplasmic absorption of the nanocrystals was observed in the form of cell niches in the secondary laminae in both animals, but in greater quantity in the samples of the treated limb. Although lamellar tissue autofluorescence was observed in both limbs, there was a wide predominance of labeled Ad-MSC in the treated limbs. <h3>Conclusion</h3> The cell labeling protocol was successful, and the labeled cells remained in the lamellar tissue of horses with chronic laminitis 24 hours after the RLP. The preliminary findings of this study indicate that the cells remained in the limb that received the RLP, demonstrating therapeutic potential in the use of this technique.

High-Efficiency Cationic Labeling Algorithm of Macroaggregated Albumin with 68Gallium.

The generator product radionuclide gallium-68(68Ga) is widely used for PET/CT imaging agents and the 68Ga-labeled MAA is an attractive alternative to 99mTc-labeled MAA. Using a commercially available MAA labeling kit for 99mTc, we presented a reliable synthesis protocol with a highly efficient, organic solvent-free cationic method in GMP conditions in the Scintomics automated synthesis unit. The labeling process was performed by incubating for 7 min at 90 °C in the borax vial containing the generator product 68GaCl3 MAA-HEPES eluted from the PSH+ cartridge with 1.5 mL 5 molar NaCl. Quality control of the final product content was examined, and radiopharmaceutical production was carried out in accordance with GMP guidelines. 68Ga eluted from the generator was obtained in more than 99% radiochemical purity and efficiency. In this case, the labeling efficiency was found to be >99%. When the results of SEM-EDX analysis in the final product were examined, it was determined that most of toxic metals were no appreciable in the product content. The radiochemical and chemical purity of the final product allows direct use without purification steps to remove "free 68Ga" or other toxic compounds.

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E.coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.