Intense Labeling Research Articles

Relieving the burden of intensive labeling for stress monitoring in the wild by using semi-supervised learning.

Stress, a natural process affecting individuals' wellbeing, has a profound impact on overall quality of life. Researchers from diverse fields employ various technologies and methodologies to investigate it and alleviate the negative effects of this phenomenon. Wearable devices, such as smart bands, capture physiological data, including heart rate variability, motions, and electrodermal activity, enabling stress level monitoring through machine learning models. However, labeling data for model accuracy assessment poses a significant challenge in stress-related research due to incomplete or inaccurate labels provided by individuals in their daily lives. To address this labeling predicament, our study proposes implementing Semi-Supervised Learning (SSL) models. Through comparisons with deep learning-based supervised models and clustering-based unsupervised models, we evaluate the performance of our SSL models. Our experiments show that our SSL models achieve 77% accuracy with a classifier trained on an augmented dataset prepared using the label propagation (LP) algorithm. Additionally, our deep autoencoder network achieves 76% accuracy. These results highlight the superiority of SSL models over unsupervised learning techniques and their comparable performance to supervised learning models, even with limited labeled data. By relieving the burden of labeling in daily life stress recognition, our study advances stress-related research, recognizing stress as a natural process rather than a disease. This facilitates the development of more efficient and accurate stress monitoring methods in the wild.

Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization

PurposeRadiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood.MethodsWe analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells.ResultsNBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells.ConclusionsOur findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.Graphical

Transition to Chronic Fibrosis in an Animal Model of Retinal Detachment With Features of Proliferative Vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the most common cause of failure of surgically repaired rhegmatogenous retinal detachment (RRD). Chemically induced and cell injection PVR models do not fully simulate the clinical characteristics of PVR in the post-RRD context. There is an unmet need for translational models in which to study mechanisms and treatments specific to RRD-PVR. RRD was induced in adult Dutch Belted rabbits. Posterior segments were fixed or processed for RNA sequencing at 6 hours and 2, 7, 14, and 35 days after induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein, alpha smooth muscle actin, vascular endothelial growth factor receptor 2, CD68, and RPE 65 kDa protein were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. Acute histopathological changes included intravitreal and intraretinal hemorrhage, leukocytic vitritis, chorioretinitis, and retinal rarefaction. Chronic lesions showed retinal atrophy, gliosis, fibrotic subretinal membranes, and epiretinal fibrovascular proliferation. Fibrillar collagen was present in the fibrocellular and fibrovascular membranes in chronic lesions. Moderate to strong labeling of glia and vasculature was detected in chronic lesions. At day 14, most cells profiled by single cell sequencing were identified as Mϋller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes was upregulated in chronic lesions. Histological and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intraretinal and periretinal fibrosis. This animal model of RRD with features of PVR will enable further research on targeted treatment interventions.

Extravascular Factor IX Pool Fed By Prophylaxis Is a True Hemostatic Barrier Against Bleeding

Factor IX (FIX) can bind to type-IV collagen in the endothelial basement membrane and diffuse into extravascular spaces. Previous studies in rodents have reported a large biodistribution of FIX. However, the potential hemostatic activity of extravascular FIX and its role in protecting against joint bleeds have not been demonstrated. The capacity of 4 different FIX molecules (plasma derived [pd] and recombinants) to bind type I and type IV collagen was studied here. FIX molecules were also administered intravenously at doses of 50 to 3000 IU/kg in FIX knock-out (KO) mice. A specific FIX signal was detected in immunohistochemistry in the liver as well as in muscles and knee joints with recombinant FIX molecules injected at 1000 and 3000 IU/kg but not at the usual clinical doses of 50-100 IU/kg, while pdFIX generated a FIX signal at all doses including 50 IU/kg. After five 100 IU/kg daily infusions of rFIX, a clear accumulation of FIX was seen in the liver and joints of animals having received 5-day daily FIX infusions with all FIX molecules, with a higher FIX labelling intensity compared to that observed after a single infusion of FIX 100 IU/kg or a single infusion of FIX 500 IU/kg. The extravascular procoagulant activity of FIX was assessed in saphenous vein bleeding assays performed 7 days after a single infusion of FIX 100 IU/kg. An additional group of FIX KO mice received intravenously for 5 days a single daily dose of 100 IU/Kg of one the four types of FIX concentrate in order to mimic prophylaxis. The number of clots formed in 15 minutes was significantly greater in mice having received prophylaxis during 5 days with 100 IU/kg FIX compared to untreated FIX KO mice (p=0.009) and to mice having received a single infusion of FIX 100 IU/kg (p<0.0001) (Figure 1). Taken together, these results show that in individuals with severe hemophilia B receiving regular prophylaxis with FIX, extravascular accumulation of FIX over time may have a significant impact on the coagulation capacity and protection towards bleeding.

Canine urothelial carcinoma: expression of Periostin in spontaneous canine urothelial carcinoma and its correlation with histological features

The tumor microenvironment is considered one of the main players in cancer development and progression and may influence the behavior of cancer cells. Periostin (POSTN) is an extracellular matrix protein, and its main functions are induction of fibrillogenesis, fibroblastic cell proliferation and migration, enhancing regeneration in normal tissue, and promoting metastasis in case of neoplasia. POSTN has already been studied in humans in several normal tissues, inflammatory processes, and neoplasms, revealing an important role in tumor progression in various types of cancer, such as colon, lung, head and neck, breast, ovarian, and prostate. In these latter, high levels of POSTN are usually associated with a more aggressive tumor behavior, tumor advanced stages, and poor prognosis, while in human bladder urothelial carcinoma (BUC), unlike in most tumors, POSTN expression seems to be downregulated. The expression of this marker has been poorly investigated in veterinary medicine; thus, this study aimed to immunohistochemically investigate the presence and the intensity of POSTN expression in canine BUCs and to determine a possible relationship between POSTN expression and histopathological features such as mitotic count and muscular and vascular invasions. For the present retrospective study, archived samples from 45 canine BUCs and 6 non-neoplastic canine bladders were considered for histological evaluation and immunohistochemical examination for the expression of POSTN. POSTN expression was semi-quantitatively assessed considering both the percentage of the neoplastic stroma positive for POSTN and the intensity of the immunohistochemical labeling. Histologically, 38 out of 45 tumors were papillary and 7 out of 45 were non-papillary. All tumors were infiltrating, being that 21 were muscle-invasive, and a significant correlation between this feature and vascular invasion emerged (P = 0.0001). In normal bladder tissue, as reported in humans, a thick and strongly positive belt of POSTN was visible, and in canine BUCs, stating that the expression is comparable with human benign as well as malignant bladder tissue, a general decrease in POSTN expression was observed except for a strongly labeled ring of POSTN observed around some neoplastic nodules infiltrating the muscle layer. Moreover, POSTN expression and mitotic count were significatively inversely correlated (P = 0.0015). The fact that POSTN protein is less expressed in urothelial carcinomas than in the normal bladder supports what was reported in human BUCs and, together with the negative correlation between mitotic count and protein expression that emerged in the present retrospective study, encourages further prospective follow-up studies to verify the possible role of POSTN in canine BUCs as a prognostic marker, and also as a possible target for the development of future anticancer therapies.

An electronic nose based on adaptive fusion of transformer-ELM with active temperature modulation algorithm for accurate odor detection in refrigerators

Accurate detection of food spoilage in refrigerators is crucial for ensuring food freshness and safety. However, due to the wide variety of gases emitted by decaying food and their uneven distribution of gases within the refrigerator, current mixed odor detection methods are not satisfactory. This study proposes a dedicated algorithm for a refrigerator electronic nose that enables precise classification of mixed food odors and prediction of their intensity. To achieve this objective, a dataset of food odor samples was collected from refrigerators, and sensory identification as well as gas chromatography-mass spectrometry analysis were performed to obtain freshness and intensity labels. The developed electronic nose algorithm incorporates key techniques, including active temperature modulation and an adaptive fusion model of lightweight Transformer-ELM, to enhance sensitivity, selectivity, and global modeling capabilities for identifying abnormal odors in volatile compounds of mixed gases. Experimental evaluations on a large-scale dataset demonstrate the effectiveness of the proposed method in classifying refrigerated food freshness and predicting odor intensity. This research contributes to the field of electronic nose technology and has potential for applications beyond refrigerator odor detection.

Rapid SERS Detection of Botulinum Neurotoxin Type A.

Surface-enhanced Raman scattering (SERS) is a powerful technique for decoding of 2-5-component mixes of analytes. Low concentrations of analytes and complex biological media are usually non-decodable with SERS. Recognition molecules, such as antibodies and aptamers, provide an opportunity for a specific binding of ultra-low contents of analyte dissolved in complex biological media. Different approaches have been proposed to provide changes in SERS intensity of an external label upon binding of ultra-low contents of the analytes. In this paper, we propose a SERS-based sensor for the rapid and sensitive detection of botulinum toxin type A. The silver nanoisland SERS substrate was functionalized using an aptamer conjugated with a Raman label. The binding of the target affects the orientation of the label, providing changes in an analytical signal. This trick allowed detecting botulinum toxin type A in a one-stage manner without additional staining with a monotonous dose dependence and a limit of detection of 2.4 ng/mL. The proposed sensor architecture is consistent with the multiarray detection systems for multiplex analyses.

Three-dimensional multi-color optical nanoscopy at sub-10-nm resolution based on small-molecule organic probes

Achieving nanometer-scale resolution remains challenging in expansion microscopy due to photon loss. To address this concern, here we develop a multi-color expansion stimulated emission depletion technique based on small-molecule probes to realize high labeling density and intensity. Our method substantially lowers the barrier to visualizing diverse intracellular proteins and their interactions in three dimensions. It enables us to achieve sub-10-nm resolution in structures such as microfilaments, lysosomes, and mitochondria, providing new insights into cell biology.

DLATA: Deep Learning-Assisted transformation alignment of 2D brain slice histology

Accurate alignment of brain slices is crucial for the classification of neuron populations by brain region, and for quantitative analysis in in vitro brain studies. Current semi-automated alignment workflows require labor intensive labeling of feature points on each slice image, which is time-consuming. To speed up the process in large-scale studies, we propose a method called Deep Learning-Assisted Transformation Alignment (DLATA), which uses deep learning to automatically identify feature points in images after training on a few labeled samples. DLATA only requires approximately 10% of the sample size of other semi-automated alignment workflows. Following feature point recognition, local weighted mean method is used as a geometrical transformation to align slice images for registration, achieving better results with about 4 fewer pixels of error than other semi-automated alignment workflows. DLATA can be retrained and successfully applied to the alignment of other biological tissue slices with different stains, including the typically challenging fluorescent stains. Reference codes and trained models for Nissl-stained coronal brain slices of mice can be found at https://github.com/ALIGNMENT2023/DLATA.

Immunohistochemical characterization of clear cell variant of hepatocellular carcinoma in a sheep

The cadaver and viscera of a mature female sheep (Ovis aries) underwent routine abattoir meat inspection. The liver was expanded by an infiltrative neoplastic mass comprising multifocal to coalescing, well-demarcated, pink to white–yellow nodules, up to 25 mm in diameter. An unencapsulated, moderately densely cellular, infiltrative neoplasm was present within the hepatic parenchyma. The neoplastic cells were arranged in solid sheets and acini supported by a moderately fine collagenous vascularized stroma. The neoplastic cells were moderately sized and polygonal, with clearly delineated cell borders and a moderate amount of cytoplasm that was clear or exhibited either globular eosinophilic deposits or fine fibrillar eosinophilic strands. The neoplastic cell nuclei were round and centrally located. The chromatin was lightly stippled and there was frequently a single, prominent, basophilic nucleolus. There were eight mitoses in 10 high-power fields (2.37 mm2). Most of the neoplastic cells had intense cytoplasmic immunolabelling for arginase 1, with frequent concurrent nuclear positivity, and mild to moderately intense punctate cytoplasmic labelling for hepatocyte specific antigen (Hep Par-1). The neoplastic cells did not label with anti-cytokeratin 19 antibody. Based on the histological appearance and the immunolabelling pattern, the neoplasm was diagnosed as the clear cell variant of a hepatocellular carcinoma.

Near Real-Time Flood Mapping with Weakly Supervised Machine Learning

Advances in deep learning and computer vision are making significant contributions to flood mapping, particularly when integrated with remotely sensed data. Although existing supervised methods, especially deep convolutional neural networks, have proved to be effective, they require intensive manual labeling of flooded pixels to train a multi-layer deep neural network that learns abstract semantic features of the input data. This research introduces a novel weakly supervised approach for pixel-wise flood mapping by leveraging multi-temporal remote sensing imagery and image processing techniques (e.g., Normalized Difference Water Index and edge detection) to create weakly labeled data. Using these weakly labeled data, a bi-temporal U-Net model is then proposed and trained for flood detection without the need for time-consuming and labor-intensive human annotations. Using floods from Hurricanes Florence and Harvey as case studies, we evaluated the performance of the proposed bi-temporal U-Net model and baseline models, such as decision tree, random forest, gradient boost, and adaptive boosting classifiers. To assess the effectiveness of our approach, we conducted a comprehensive assessment that (1) covered multiple test sites with varying degrees of urbanization, and (2) utilized both bi-temporal (i.e., pre- and post-flood) and uni-temporal (i.e., only post-flood) input. The experimental results showed that the proposed framework of weakly labeled data generation and the bi-temporal U-Net could produce near real-time urban flood maps with consistently high precision, recall, f1 score, IoU score, and overall accuracy compared with baseline machine learning algorithms.

Type II Diabetes Mellitus Causes Extracellular Matrix Alterations in the Posterior Cornea That Increase Graft Thickness and Rigidity.

There is a pressing need to investigate the impact of type II diabetes mellitus on the posterior cornea in donor tissues given its increasing prevalence and potential impact on endothelial keratoplasty surgical outcomes. Immortalized human cultured corneal endothelial cells (CECs; HCEC-B4G12) were grown in hyperglycemic media for 2 weeks. Extracellular matrix (ECM) adhesive glycoprotein expression and advanced glycation end products (AGEs) in cultured cells and corneoscleral donor tissues, as well as the elastic modulus for the Descemet membrane (DMs) and CECs of diabetic and nondiabetic donor corneas, were measured. In CEC cultures, increasing hyperglycemia resulted in increased transforming growth factor beta-induced (TGFBI) protein expression and colocalization with AGEs in the ECM. In donor corneas, the thicknesses of the DM and the interfacial matrix (IFM) between the DM and stroma both increased from 8.42 ± 1.35 µm and 0.504 ± 0.13 µm in normal corneas, respectively, to 11.13 ± 2.91 µm (DM) and 0.681 ± 0.24 µm (IFM) in non-advanced diabetes (P = 0.013 and P = 0.075, respectively) and 11.31 ± 1.76 µm (DM) and 0.744 ± 0.18 µm (IFM) in advanced diabetes (AD; P = 0.0002 and P = 0.003, respectively). Immunofluorescence in AD tissues versus controls showed increased AGEs (P < 0.001) and markedly increased labeling intensity for adhesive glycoproteins, including TGFBI, that colocalized with AGEs. The elastic modulus significantly increased between AD and control tissues for the DMs (P < 0.0001) and CECs (P < 0.0001). Diabetes and hyperglycemia alter human CEC ECM structure and composition, likely contributing to previously documented complications of endothelial keratoplasty using diabetic donor tissue, including tearing during graft preparation and reduced graft survival. AGE accumulation in the DM and IFM may be a useful biomarker for determining diabetic impact on posterior corneal tissue.

Enhancement of Scaffold In Vivo Biodegradability for Bone Regeneration Using P28 Peptide Formulations.

The field of bone tissue engineering has shown a great variety of bone graft substitute materials under development to date, with the aim to reconstruct new bone tissue while maintaining characteristics close to the native bone. Currently, insufficient scaffold degradation remains the critical limitation for the success of tailoring the bone formation turnover rate. This study examines novel scaffold formulations to improve the degradation rate in vivo, utilising chitosan (CS), hydroxyapatite (HAp) and fluorapatite (FAp) at different ratios. Previously, the P28 peptide was reported to present similar, if not better performance in new bone production to its native protein, bone morphogenetic protein-2 (BMP-2), in promoting osteogenesis in vivo. Therefore, various P28 concentrations were incorporated into the CS/HAp/FAp scaffolds for implantation in vivo. H&E staining shows minimal scaffold traces in most of the defects induced after eight weeks, showing the enhanced biodegradability of the scaffolds in vivo. The HE stain highlighted the thickened periosteum indicating a new bone formation in the scaffolds, where CS/HAp/FAp/P28 75 µg and CS/HAp/FAp/P28 150 µg showed the cortical and trabecular thickening. CS/HAp/FAp 1:1 P28 150 µg scaffolds showed a higher intensity of calcein green label with the absence of xylenol orange label, which indicates that mineralisation and remodelling was not ongoing four days prior to sacrifice. Conversely, double labelling was observed in the CS/HAp/FAp 1:1 P28 25 µg and CS/HAp/FAp/P28 75 µg, which indicates continued mineralisation at days ten and four prior to sacrifice. Based on the HE and fluorochrome label, CS/HAp/FAp 1:1 with P28 peptides presented a consistent positive osteoinduction following the implantation in the femoral condyle defects. These results show the ability of this tailored formulation to improve the scaffold degradation for bone regeneration and present a cost-effective alternative to BMP-2.

Study of the cell proliferation index (Ki67) in inflammatory odontogenic cysts

IntroductionInflammatory odontogenic cysts are lesions that develop from the cellular remains of odontogenesis. They are lesions that may recur after incomplete surgical enucleation with a variable clinical appearance depending on the clinical form. These clinical variations could be explained by the biological behaviour of the lesion cells which can be explored by the study of the cell proliferation index (Ki67). This justifies the initiation of this work whose objective was to study the level of expression of Ki67 and to measure the index of proliferation in odontogenic inflammatory cysts. Material and methodThis was a cross-sectional study with an analytical aim on surgical parts of odontogenic inflammatory cysts over a period of 31 months in the odontostomatology department of the Hôpital Général Idrissa Pouye. The Ki67 immunohistochemistry study was performed on 3μ thick histological sections of paraffin-fixed tissue. The inclusion criteria were any surgical specimen of odontogenic inflammatory cyst that we could collect. The variables studied were sociodemographic, clinical and histopathological. ResultsRadiculo-dental cysts represented 80.5% of the surgical specimens collected and residual cysts 12.2%. The percentage of Ki67-labelled cells was greater than 15% in 80.5% of the odontogenic inflammatory cysts and less than 7% in 19.5% of the cases. In addition, intense labelling was noted in radiculo-dental cysts and weak labelling in the other clinical forms of inflammatory odontogenic cysts obtained. An association was noted between the percentage of Ki67 labelled cells and histological types (p-value <0.001); but also between the intensity of Ki67 labelling and histological types (p-value <0.001). ConclusionThe cell proliferation index (Ki67) could be used as a prognostic biomarker in odontogenic inflammatory cysts.

A new Drosophila melanogaster fly that expresses GFP-tagged Orco

Insects perceive countless odors with the help of odorant receptors (ORs) expressed by olfactory sensory neurons (OSNs). OR complexes are composed of odor-specific proteins OrX and olfactory co-receptor proteins Orco, with both extracellular C-terminus and intracellular N-terminus. Here, we generated a genetically modified Drosophila melanogaster fly line that expresses Orco proteins with an N-terminally fused green fluorescent protein (GFP). We generated four genotypes (homozygous and heterozygous) for which we performed antennal cryosectioning and immunolabeling to determine which genotype would show high GFP labeling intensity in the dendritic areas of OSNs. We found that fluorescent intensity was the highest in homozygous flies. As Orco is known to guide OrX proteins to the OSN dendrites, we checked whether the fusion of GFP to the Orco N-terminus may affect such trafficking and thereby OR function. Using single sensillum recording (SSR), we found no difference in the olfactory capability of homozygous flies compared with wild-type flies. As future application, we want to use this transgenic fly line to resolve the structure of OR complexes using Cryo electron microscopy (cryo-EM).

Neuronal muscarinic acetylcholine receptor subtype modulation of inspiratory bursting at hypoglossal motoneurons in developing neonatal mice in vitro

Muscarinic acetylcholine receptor modulation contributes to changes in hypoglossal motoneuron excitability through pre- and post-synaptic effects, an important mechanism for regulation of airway tone during sleep. Previous data indicate a net excitatory effect of muscarinic modulation of inspiratory bursting in neonatal mouse brainstem slices, yet a net inhibitory effect on inspiratory bursting in adult rats. We tested the hypothesis that a developmental shift in muscarinic modulation impacts hypoglossal motoneurons, using neuroanatomical and electrophysiological methods. Immunofluorescence confirmed hypoglossal motoneurons express M1, M2, M3, and M5 muscarinic subtypes. We observed maturational decreases in M1 and M5 labeling intensity, M3 labeling intensity generally decreased with postnatal (P) age, although there was a transient increase in expression at P17. M2 labeling intensity did not change, although distribution of M2 labeling with postnatal maturation may change. Next, using the in-vitro rhythmic slice preparation from CD1 mice of either sex, local application of muscarine (100 μM) into the hypoglossal nucleus was tested in mice P0-14. The net effect of applying muscarine results in excitatory output after local application into the rhythmic slice because each data point is a positive percent change from control. The magnitude of this potentiation appears to lessen as the mice age toward P14. To evaluate the mechanisms through which the magnitude of muscarinic potentiation of inspiratory bursting changes with postnatal maturation, we next assessed the role of excitatory and inhibitory muscarinic receptor subtypes. Blocking M1 locally (pirenzepine, 100 μM) significantly decreased muscarine-mediated potentiation of inspiratory bursting (to 72±14% of control muscarine response, n=9, P0-5). Preliminary data indicate bath application of 4-DAMP (M3 antagonist) attenuated muscarinic potentiation of inspiratory bursting (range: to 7%-25% of control burst response, n=3, P0-5). Modulating M5 locally with a positive allosteric modulator VU 0238429 (1000 μM) had little effect on burst amplitude (119±4% of control burst response, n=3, P0-6). Blocking M2 locally (methoctramine, 2μM) showed no effect on inspiratory burst potentiation after applying muscarine (181±65% vs 187±69%, n=7, P0-5). Bath application of methoctramine did not have a significant effect on inspiratory burst potentiation by muscarine (179±65% vs control 182±90%, n=10) in P0-5 mice while preliminary analysis suggests an increasing effect of blocking M2 with postnatal maturation (147±56% vs control 134±30%, n=4, P9-14). These data support excitatory muscarinic receptor subtypes contributing to the excitatory response early in postnatal maturation. Based on our neuroanatomical data, we speculate the increasing inhibitory response to muscarinic modulation with postnatal maturation is due to a decreased contribution of excitatory muscarinic receptor subtypes. NIH R15HL148870 (Revill) and NIH/NHLBI R25 HL126140 (Moreno, Garcia, Parthasarathy) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EXPRESSION OF CYCLIN D1 IN ORAL SQUAMOUS CELL CARCINOMA AND ITS CORRELATION WITH HISTOPATHOLOGICAL DIFFERENTIATION.

BACKGROUND: Cyclin D1 is an important protein that has a widespread role in governing cell proliferation rates, and cell cycle regulations. In variety of tumors overexpression of Cyclin D1 has been reported. Present study was carried out to study the expression of cyclin D1 in oral squamous cell carcinoma and its association with histopathological differentiation and staging. To correlate expression AIM &amp; OBJECTIVES: of cyclin D1 with histological grading and staging in oral squamous cell carcinoma. 98 biopsy specimens of oral MATERIALS &amp; METHODS: squamous cell carcinoma were included in this study and were immunohistochemically evaluated for expression of cyclin D1. Samples were received and reported in Department of Pathology Gandhi Medical College, Bhopal, Madhya Pradesh A total of 98 cases of oral RESULT: squamous cell carcinoma were studied. Cyclin D1 reactivity was seen in 76% cases of oral squamous cell carcinoma. Cyclin D1 reactivity did not correlate with site, tumor grade, and stage, whereas Cyclin D1 expression score which was obtained by multiplication of labeling index score and intensity showed signicant correlation with tumor grade and stage. Higher Cycli CONCLUSION: n D1 expression observed in patients with less differentiated tumours and with higher stage. Therefore, there is value of analysing cyclin D1 amplication and expression, for prognostic evaluation. This may also be further used for targeted therapy in oral squamous cell carcinoma

The emerging role of lysine succinylation in ovarian aging

BackgroundOvarian aging is a process of decline in its reserve leading to ovary dysfunction and even reduced health quality in offspring. However, aging-related molecular pathways in the ovary remain obscure. Lysine succinylation (Ksuc), a newly post-translational modification (PTM), has been found to be broadly conserved in both eukaryotic and prokaryotic cells, and associated with multiple pathophysiological processes. There are no relevant reports revealing a link between the molecular mechanisms of ovarian aging and Ksuc.MethodsThe level of Ksuc in ovaries of aged and premature ovarian insufficiency (POI) mice were detected by immunoblotting and immunohistochemical. To further explore the role of Ksuc in ovarian aging, using in vitro mouse ovary tissue culture and an in vivo mouse model with changed Ksuc level.ResultsIncreased Ksuc in ovaries of aged and POI mice and distribution of Ksuc in various types of mice ovarian cells and the high level of Ksuc in granulosa cells (GCs) were revealed. Histological assessments and hormone levels analyses showed that the high Ksuc level down-regulated the ovarian index and the anti-Müllerian hormone (AMH) and estrogen levels, and increased follicular atresia. Moreover, in the high Ksuc groups, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) intensities and the expression of Cleaved-caspase-3 increased and the expression of B-cell lymphoma-2 (Bcl-2) decreased together with positively-expressed P21, an aging-related marker. These results suggest that ovarian aging is likely associated with alteration in Ksuc.ConclusionThe present study has identified Ksuc in mouse ovary and found that high Ksuc level most likely contributes to ovarian aging which is expected further investigation to provide new information for delaying physiological ovarian aging and treating pathological ovarian aging.

Abstract 4350: Pharmacodynamic biomarkers: Evaluation of oncology drug target engagement in human plucked scalp and beard hair

Abstract We have developed a method to assess target expression changes following exposure of plucked hairs to chemotherapeutic agents and other potential therapeutics. Successful ex vivo responses provide proof of concept data prior to clinical studies. Plucked hair is a valuable surrogate biomarker tissue to monitor pharmacodynamic (PD) responses in a clinical trial. The collection of hairs is minimally invasive, simple and is amenable to frequent sampling. Since hairs are epithelial appendages, many signaling pathways active in other epithelial tissues, including cancers, are also present in hairs. Highly proliferative plucked human scalp hair can be utilized as a surrogate tissue to measure proliferation, phosphorylation and DNA damage responses after treatment. Furthermore, hairs are highly vascularized, suggesting that administered Test Articles may be delivered efficiently to the hair in a similar manner to other highly vascularized tissues, including many tumor types. Human plucked hair (either from the scalp or beard) was placed in maintenance media in the presence of conventional or targeted chemotherapeutic agents, known for their different mechanisms of action (e.g. Gemcitabine, Carboplatin, Tarceva), for a range of time points. Hairs were then fixed post-exposure and longitudinal sections immunohistochemically labelled for various markers including p-ERK1/2, p-AKT, p-Chk1, g-H2AX, Ki67 and androgen receptor. Quantitative image analysis to measure the level of labelling was performed using an Aperio ScanScope. The relative number of positively labelled cell nuclei, or relative tissue area (depending on the labelling pattern of the target), of the hair outer root sheath labelled was analyzed, along with label intensity. In subsequent trial patients, formaldehyde fixed plucked hairs were returned to the lab, similarly sectioned, labelled and quantified. In addition, where antibody labelling is not possible or more detailed information on the modulation of a pathway may be useful, drug treated plucked hairs can be subjected to Next Generation Sequencing (NGS) gene expression analysis. The expected changes in labelling were observed. For example, Gemcitabine, which is linked to DNA polymerase inhibition, increased p-Chk1 labelling 4-fold after 4-8 hours and strongly induced p53 by 24 hours. Tarceva, an inhibitor of the EGF pathway, decreased levels of both p-ERK1/2 and p-AKT 3-fold after only 10 minutes. The alkylating agent carboplatin increased g-H2AX labelling up to 12-fold after 24 hours. We have demonstrated that plucked hair is a valuable PD biomarker tissue for various chemotherapy agents. Proof of concept studies performed ex vivo can inform the design of clinical validation studies by indicating optimum markers and timepoints. Further, each hair is an independent unit thereby allowing independent replicate tissues (hairs) to be sampled. Citation Format: Greg Tudor, Frida Ponthan, Adam Boanas, Aude-Marine Bonavita, Catherine Booth. Pharmacodynamic biomarkers: Evaluation of oncology drug target engagement in human plucked scalp and beard hair. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4350.

Pendrin abundance, subcellular distribution, and function are unaffected by either αENaC gene ablation or by increasing ENaC channel activity

The intercalated cell Cl−/HCO3− exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl− absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle’s syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle’s variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle’s mutation increased total Cl− absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl− absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.