Epithelial Growth Research Articles

Hippo cooperates with p53 to maintain foregut homeostasis and suppress the malignant transformation of foregut basal progenitor cells

Basal progenitor cells serve as a stem cell pool to maintain the homeostasis of the epithelium of the foregut, including the esophagus and the forestomach. Aberrant genetic regulation in these cells can lead to carcinogenesis, such as squamous cell carcinoma (SCC). However, the underlying molecular mechanisms regulating the function of basal progenitor cells remain largely unknown. Here, we use mouse models to reveal that Hippo signaling is required for maintaining the homeostasis of the foregut epithelium and cooperates with p53 to repress the initiation of foregut SCC. Deletion of Mst1/2 in mice leads to epithelial overgrowth in both the esophagus and forestomach. Further molecular studies find that Mst1/2-deficiency promotes epithelial growth by enhancing basal cell proliferation in a Yes-associated protein (Yap)-dependent manner. Moreover, Mst1/2 deficiency accelerates the onset of foregut SCC in a carcinogen-induced foregut SCC mouse model, depending on Yap. Significantly, a combined deletion of Mst1/2 and p53 in basal progenitor cells sufficiently drives the initiation of foregut SCC. Therefore, our studies shed light on the collaborative role of Hippo signaling and p53 in maintaining squamous epithelial homeostasis while suppressing malignant transformation of basal stem cells within the foregut.

Effectiveness of APG and Honey Gauze in Pressure Injury of Elderly: A Randomized Control Trial.

This study was designed to evaluate the efficiency of the combination of autologous platelet-rich plasma gel (APG) and Manuka honey gauze in the treatment of Stages 3-4 pressure injury of older adults. Patients were divided into four groups: Manuka honey gauze and APG (M + A), Manuka honey gauze (M), APG (A), and a control group (C). Different treatments were given, then wound bed coverage with granulation tissue, wound size reduction, and Pressure Ulcer Scale for Healing (PUSH) score were examined. Paraffin-embedded sections of wound tissues were analyzed and wound swab cultures were assessed. Kruskal-Wallis test and Mann-Whitney U test were performed in statistical analysis at a 5% significance level. A total of 42 patients were accepted. Significant increase of wound bed coverage with granulation tissue (51.24%, P = .004, Kruskal-Wallis test) and decrease of PUSH score (-5) were observed in the M + A group at the end of the observation (P = .032, Mann-Whitney U test). The hematoxylin-eosin staining of wound tissues showed that typical squamous epithelium was seen in wound bed of patient in M + A group. Manuka honey gauze and APG were proved to be superior treatments for pressure injury of old patient. Increase of granulation tissue coverage, reduction of PUSH score, and improved growth of epithelium were observed in M + A group. There was no side-effect, and the treatment would not cause infection.

Detection of EGFR T790M mutation using liquid biopsy for non-small cell lung cancer: Utility of droplet digital polymerase chain reaction vs. cobas real-time polymerase chain reaction

BackgroundDigital platforms for mutation detection yield higher sensitivity than non-digital platforms but lack universal positive cut-off values that correlate with the outcome of osimertinib treatment. This study determined compared droplet digital polymerase chain reaction (ddPCR) to the standard cobas assay for epithelial growth factor receptor (EGFR) T790M mutation detection in patients with non-small cell lung cancer. MethodsStudy patients had EGFR-mutant tumours with disease progression on first/second generation EGFR tyrosine kinase inhibitors, and osimertinib treatment after T790M mutation detection. T790M status was tested by cobas assay using liquid biopsy, and only by ddPCR if an EGFR mutation was identified but T790M was negative. Clinical efficacy of osimertinib was compared between patients with T790M detected by cobas vs. only by ddPCR. A positive cut-off value for ddPCR was determined by assessing efficacy with osimertinib. Results61 patients had tumors with an acquired T790M mutation, 38 detected by cobas and an additional 23 only by ddPCR. The median progression-free survival (PFS) for the cobas- and ddPCR-positive groups was 9.5 and 7.8 months, respectively (p=0.43). For ddPCR, a fractional abundance (FA) of 0.1% was used as a cut-off value. The median PFS of patients with FA ≥0.1% and <0.1% was 8.3 and 4.6 months, respectively (p=0.08). FA ≥0.1% was independently associated with a longer PFS. ConclusionUsing ddPCR to follow up the cobas assay yielded more cases (38% of total) with a T790M mutation. A cut-off value of FA ≥0.1% identified patients who responded as well to osimertinib as those identified by cobas assay. This sequential approach should detect additional patients who might benefit from osimertinib treatment.

Phospholipase D Mediates Glutamine-Induced mTORC1 Activation to Promote Porcine Intestinal Epithelial Cell Proliferation

BackgroundThe intestinal epithelium is one of the fastest self-renewal tissues in the body, and glutamine plays a crucial role in providing carbon and nitrogen for biosynthesis. In intestinal homeostasis, phosphorylation-mediated signaling networks that cause altered cell proliferation, differentiation, and metabolic regulation have been observed. However, our understanding of how glutamine affects protein phosphorylation in the intestinal epithelium is limited, and identifying the essential signaling pathways involved in regulating intestinal epithelial cell growth is particularly challenging. ObjectivesThis study aimed to identify the essential proteins and signaling pathways involved in glutamine’s promotion of porcine intestinal epithelial cell proliferation. MethodsPhosphoproteomics was applied to describe the protein phosphorylation landscape under glutamine treatment. Kinase-substrate enrichment analysis was subjected to predict kinase activity and validated by qRT-PCR and Western blotting. Cell Counting Kit-8, glutamine rescue experiment, chloroquine treatment, and 5-fluoro-2-indolyl deschlorohalopemide inhibition assay revealed the possible underlying mechanism of glutamine promoting porcine intestinal epithelial cell proliferation. ResultsIn this study, glutamine starvation was found to significantly suppress the proliferation of intestinal epithelial cells and change phosphoproteomic profiles with 575 downregulated sites and 321 upregulated sites. Interestingly, phosphorylation of eukaryotic initiation factor 4E-binding protein 1 at position Threonine70 was decreased, which is a crucial downstream of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Further studies showed that glutamine supplementation rescued cell proliferation and mTORC1 activity, dependent on lysosomal function and phospholipase D activation. ConclusionIn conclusion, glutamine activates mTORC1 signaling dependent on phospholipase D and a functional lysosome to promote intestinal epithelial cell proliferation. This discovery provides new insight into regulating the homeostasis of the intestinal epithelium, particularly in pig production.

The Drosophila tumour suppressor Lgl and Vap33 activate the Hippo pathway through a dual mechanism.

The tumour suppressor, Lethal (2) giant larvae [Lgl; also known as L(2)gl], is an evolutionarily conserved protein that was discovered in the vinegar fly Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity. Lgl links cell polarity and tissue growth through regulation of the Notch and the Hippo signalling pathways. Lgl regulates the Notch pathway by inhibiting V-ATPase activity via Vap33. How Lgl regulates the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Vap33 physically and genetically interacts with the actin cytoskeletal regulators RtGEF (Pix) and Git, which also bind to the Hippo protein (Hpo) and are involved in the activation of the Hippo pathway. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether, our data suggest that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism: (1) through interaction with RtGEF, Git and Arf79F, and (2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.

Durable response to first-line treatment with AZD3759 (zorifertinib) in a patient with epithelial growth factor receptor mutated non-small cell lung cancer and untreated multiple brain metastasis

Central nervous system (CNS) metastases, including brain and leptomeningeal metastasis, are associated with poor prognosis in epidermal growth factor receptor (&lt;i&gt;EGFR&lt;/i&gt;)-mutated non-small cell lung cancer (NSCLC) patients. CNS metastasis occurs in more than 50% of &lt;i&gt;EGFR&lt;/i&gt;-mutated NSCLC cases during the treatment course. The treatment options for CNS metastases in these patients are limited, and there have been ongoing efforts to develop targeted agents with enhanced CNS penetration. AZD3759 (zorifertinib) is a novel &lt;i&gt;EGFR&lt;/i&gt; tyrosine kinase inhibitor with a high capability of CNS penetration and proven efficacy with tolerable safety profiles. In this report, we describe a case of a patient with &lt;i&gt;EGFR&lt;/i&gt;-mutant NSCLC that was initially diagnosed with multiple brain metastasis who demonstrated prolonged response of both intra- and extracranial lesions over 7 years with AZD3759 as first-line treatment.

Comparative analysis of the cytoarchitecture of the excretory bladder of adult Digenea (Platyhelminthes) with consideration of the presence of mineralized excretory corpuscles in marine and freshwater adult worms

The ultrastructural differences are shown between the cytoarchitecture of the excretory bladder and excretory inclusions in four digenean species, two azygiids, the marine Otodistomum cestoides and the freshwater Azygia lucii, the marine derogenid Derogenes varicus and the freshwater allocreadiid Acrolichanus auriculatus. The unusual cytoarchitecture of the bladder epithelium of the azygiid digeneans, consisting along its entire length of two alternating, morphologically different zones, tegumental and cellular excretory epithelial zones, connected by septate junctions, has recorded for the first time for the Digenea and, in general, for the Neodermata. It, possible, suggests the participation of the tegumental distal cytoplasmic layer in the formation of their excretory bladder epithelium. Like most digeneans, the excretory bladder of A. auriculatus and D. varicus has a syncytial epithelial lining. Based on available literature and our own results, we can confirm the presence of the excretory corpuscles in adult marine digeneans and their absence from freshwater species, regardless of the digenean localization in their host. The present study shown that in marine digeneans, the excretory corpuscles are associated with specialized excretory cells or excretory syncytial epithelium. Ultrastructural data were obtained on the possible growth of the excretory bladder epithelium due to the migration of undifferentiated cells into the epithelial lining in studied marine species. We may assume that the bladder epithelium of marine adult digeneans specializes, in addition to the excretory function, in osmoregulatory function.

Antibacterial properties of marine algae incorporated polylactide acid membranes as an alternative to clinically applied different collagen membranes

The reconstruction of bony defects in the alveolar crest poses challenges in dental practice. Guided tissue regeneration (GTR) and guided bone regeneration (GBR) procedures utilize barriers to promote bone regeneration and prevent epithelial growth. This study focuses on evaluating the antibacterial properties of marine algae-polylactic acid (PLA) composite membranes compared to commercially available collagen membranes. Marine algae (Corallina elongata, Galaxaura oblongata, Cystoseira compressa, Saragassum vulgare, and Stypopodium schimperi) were processed into powders and blended with PLA to fabricate composite membranes. Cytocompatibility assays using human periodontal ligament fibroblasts (n = 3) were performed to evaluate biocompatibility. Antibacterial effects were assessed through colony-forming units (CFU) and scanning electron microscopy (SEM) analysis of bacterial colonization on the membranes. The cytocompatibility assays demonstrated suitable biocompatibility of all marine algae-PLA composite membranes with human periodontal ligament fibroblasts. Antibacterial assessment revealed that Sargassum vulgare-PLA membranes exhibited the highest resistance to bacterial colonization, followed by Galaxaura oblongata-PLA and Cystoseira compressa-PLA membranes. SEM analysis confirmed these findings and revealed smooth surface textures for the marine algae-PLA membranes compared to the fibrous and porous structures of collagen membranes. Marine algae-PLA composite membranes show promising antibacterial properties and cytocompatibility for guided bone and tissue regeneration applications. Sargassum vulgare-PLA membranes demonstrated the highest resistance against bacterial colonization. These findings suggest that marine algae-PLA composite membranes could serve as effective biomaterials for infection control and tissue regeneration. Further in vivo validation and investigation of biodegradation properties are necessary to explore their clinical potential.Graphical

The risk of pocket infection or bloodstream infection following central venous port placement for the patients with Cetuximab therapy.

Cetuximab is a molecular targeted drug that targets epithelial growth factor receptors. The skin toxicity of cetuximab arising from epithelial growth factor inhibition is well known. Some patients with cetuximab therapy decided to make central venous port during the long-term intravenous treatments. Therefore, the author hypothesized that cetuximab administration might increase the risk of central venous port-related infection due to damage to skin barrier function. The main aim of the present study was to investigate the relationship between cetuximab administration and central venous port-related infection. A total of 83 patients had a central venous port placed from 2016 through 2021. We analyzed, retrospectively, the relationship between cetuximab therapy and the incidence of central venous port-related infection involving central line-associated bloodstream infection and pocket infection. Additionally, the risk factors of central venous port-related infection were examined in the population undergoing cetuximab therapy. In total populations (83 cases), central line-associated bloodstream infection happened in five patients (6%) and pocket infection happened in six patients (7%) after central venous port placement. In the cetuximab therapy group (45 cases), there were four patients with central line-associated bloodstream infection (9%) and six with pocket infection (13%). The pocket infection happened more frequently in the cetuximab group than the other group with significant differences. Additionally, in the cetuximab group, the patients who had an interval of less than seven days between central venous port placement and cetuximab dosing, or central venous port placement preceded by cetuximab dosing had more pocket infection with significant differences. Skin complications after the central venous port placement were related to cetuximab administration and the timing of cetuximab therapy.

Increased expression of SSEA-4 on TKI-resistant non–small cell lung cancer with EGFR-T790M mutation

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme β3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Potential urinary biomarkers in preeclampsia: a narrative review.

Preeclampsia (PE) is a highly relevant pregnancy-related disorder. An early and accurate diagnosis is crucial to prevent major maternal and neonatal complications and mortality. Due to the association of kidney dysfunction with the pathophysiology of the disease, urine samples have the potential to provide biomarkers for PE prediction, being minimally invasive and easy to perform. Therefore, searching for novel biomarkers may improve outcomes. This narrative review aimed to summarize the scientific literature about the traditional and potential urinary biomarkers in PE and to investigate their applicability to screen and diagnose the disorder. A non-systematic search was performed in PubMed/MEDLINE, Scopus, and SciELO databases. There is significant divergence in the literature regarding traditionally used serum markers creatinine, cystatin C, and albuminuria, accuracy in PE prediction. As for the potential renal biomarkers investigated, including vascular epithelial growth factor (VEGF), placental growth factor (PlGF), and soluble fms-like tyrosine kinase (sFlt-1), urinary levels of PlGF and sFtl-1/PlGF ratio in urine seem to be the most promising as screening tests. The assessment of the global load of misfolded proteins through urinary congophilia, podocyturia, and nephrinuria has also shown potential for screening and diagnosis. Studies regarding the use of proteomics and metabolomics have shown good accuracy, sensitivity, and specificity for predicting the development and severity of PE. However, there are still many divergences in the literature, which requires future and more conclusive research to confirm the predictive role of urinary biomarkers in pregnant women with PE.

Refining first-line treatment decision in RAS wildtype (RAS-WT) metastatic colorectal cancer (mCRC) by combining clinical biomarkers: Results of the randomized phase 3 trial FIRE-3 (AIO KRK0306).

13 Background: Optimal patient selection for first-line treatment targeting epithelial growth factor receptor (EGFR) in RAS-WT mCRC is based on primary tumor sidedness (PTS) with anti-EGFR being the preferred option for patients with left-sided mCRC (LC). Right-sided mCRCs (RC) are preferentially treated in combination with bevacizumab targeting vascular endothelial growth factor (VEGF). Here, improvement in patient selection was evaluated by combining clinical biomarkers beyond PTS using the randomized phase III trial FIRE-3. Methods: FIRE-3 evaluated first-line FOLFIRI (folinic acid, fluorouracil and irinotecan) plus cetuximab (FOLFIRI/Cet) versus FOLFIRI plus bevacizumab (FOLFIRI/Bev) in patients with RAS-WT mCRC. Besides PTS, further clinical biomarkers were evaluated in pairwise combinations using Cox regression models and model-based recursive partitioning with Weibull models to predict treatment benefit of either treatment arm regarding overall survival (OS): age, sex, liver-limited disease status (LLD) and baseline carcinoembryonic antigen serum level (CEA). The resulting P-values of second-order interactions were adjusted using Holm-Bonferroni correction. The model with the best test statistics and P-value was chosen for further evaluations. Results: In 400 patients with RAS-WT mCRC, a model combining PTS and LLD status best predicted treatment outcome of either treatment arm (c-index = 0.603, p=0.005). Here, a significant survival benefit of FOLFIRI/Cet over FOLFIRI/Bev was evident in patients with LC/non-LLD (HR 0.62, p=0.02) compared to LC/LLD (HR 0.83, p=0.40). In patients with RC, FOLFIRI/Bev was significantly associated with increased OS compared to FOLFIRI/Cet when patients suffered from non-LLD (HR 2.09, p=0.010). However, patients with RC/LLD rather had a benefit from FOLFIRI/Cet compared to FOLFIRI/Bev (HR 0.59, p=0.218). Conclusions: Combining clinical biomarkers PTS and LLD status might improve optimal patient selection for targeted first-line treatment in RAS-WT mCRC. Validation in further data sets is warranted. Clinical trial information: NCT00433927 .

Evaluation of chicken chorioallantoic membrane model for tumor imaging and drug development: Promising findings.

The chicken chorioallantoic membrane (CAM) model is a potential alternative to the mouse model based on the 3R principles. However, its value for determination of the invivo behaviors of radiolabeled peptides through positron emission tomography (PET) imaging needed investigation. Herein, the chicken CAM tumor models were established, and their feasibility was evaluated for evaluating the imaging properties of radiolabeled peptides using a 68 Ga-labeled HER2 affibody. Two human breast cancer cell lines were inoculated into chicken CAM and mice, respectively. The tumor-targeting potential and pharmacokinetic profile of a 68 Ga-labeled affibody, 68 Ga-MZHER, in both tumor models were also determined. The tumor-formation time in chicken CAM model was shorter than that of mouse model. The uptake values of human epithelial growth factor receptor-2 (HER2)-positive Bcap37 tumors in chicken CAM and mouse models were 5.36 ± 0.26% ID/g and 5.26 ± 0.43% ID/g at 30 min postinjection of 68 Ga-MZHER, respectively. At the same time points, the uptake values of HER2-negative MDA-MB-231 tumors in the chicken CAM models and mouse models were 1.57 ± 0.15% ID/g and 1.67 ± 0.25% ID/g, respectively. Exvivo biodistribution confirmed that more radioactivity accumulated in Bcap37 tumors than in MDA-MD-231 tumors in both CAM and mouse models. In this study, the CAM tumor model was successfully prepared. The chicken CAM model is a novel tool for quickly determining the invivo properties of radiolabeled peptides targeting biomarkers. It may be beneficial for early monitoring of the therapeutic effect of a new drug through PET imaging with specific peptides.

TPGS-modified Chitosan Nanoparticles of EGFR Inhibitor: Physicochemical and In vitro Evaluation against HepG2 Cell Lines.

Gefitinib (GFN) is an Epithelial Growth Factor Receptor (EGFR) inhibitor, and Food and Drug Administration (FDA) has approved medication to treat lung cancer. However, this investigation aimed to produce and characterize Gefitinib (GFN)-loaded chitosan and soy lecithin nanoparticles (NPs) modified with D-α-tocopheryl polyethylene glycol 1000 succinate mono ester (TPGS) and assess their therapeutic potential against HepG2 liver cell lines. Chitosan, a cationic polymer with biocompatible and biodegradable properties, was combined with soy lecithin to develop the NPs loaded with GFN using a self-organizing ionic interaction methodology. The entrapment efficiency and drug loading were found to be 59.04±4.63 to 87.37±3.82% and 33.46±3.76 to 49.50±4.35%, respectively, and results indicated the encapsulation of GEN in NPs. The pH of the formulations was observed between 4.48-4.62. Additionally, all the prepared NPs showed the size and PDI range of 89.2±15.9 nm to 799.2±35.8 nm and 0.179±0.065 to 0.455±0.097, respectively. The FTIR bands in optimized formulation (GFN-NP1) indicated that the drug might be contained within the NP's core. The SEM photograph revealed the spherical shape of NPs. The kinetic release model demonstrated the combination of diffusion and erosion mechanisms. The IC50 value of GFN and GFN-NP1 formulation against the HepG2 cell lines were determined and found to be 63.22±3.36 μg/ml and 45.80±2.53 μg/ml, respectively. DAPI and PI staining agents were used to detect nuclear morphology. It was observed that the optimized GFN-NP1 formulation successfully internalized and inhibited the growth of HepG2 cells. Hence, it can be concluded that the prepared NPs can be a new therapeutic option for treating liver cancer.

Human apical-out nasal organoids reveal an essential role of matrix metalloproteinases in airway epithelial differentiation

Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanisms of ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, the gel gradually degrade, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increases during organoid differentiation, where inhibition of MMPs significantly suppresses the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs is found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases.

Flap of the sternocephalicus muscle in the repair of a partial defect in the trachea of a rabbit (Oryctolagus cuniculus).

The current study aimed at evaluating the repair of a partial defect of the trachea with a muscle flap, an advanced technique that employs combined suture patterns. Sixteen healthy male New Zealand white rabbits were used as an experimental model. A partial defect in the trachea within the ventral region of the fourth to eighth tracheal ring was created. Subsequently, repair was initiated with a flap of the sternocephalicus muscle. The animals were divided into four groups for postoperative evaluation using clinical, tracheoscopic, and histopathological analyses. Each group was separated according to the time of euthanasia, programmed at interval of seven (G7), 15 (G15), 30 (G30), and 60 days (G60). One animal from the G60 group died, whereas the other animals had good surgical recovery without serious changes in the breathing pattern. The major clinical signs observed were stridor and coughing. Tracheoscopy revealed secretions in the tracheal lumen, exuberant granulation, and stenosis. Histopathological analysis showed growth of the ciliary respiratory epithelium at the flap site 30 days after implantation. Partial repair showed satisfactory results owing to the anatomical location of the muscle, adequate vascular support, and structural and physiological maintenance without serious changes in the respiratory system.

Modulation of steroid pathway to preserve corneal transparency

Purpose: Corneal neovascularization (CN) is a major cause of blindness worldwide. We previously showed that endothelial mineralocorticoid receptor (MR) contributed to CN in mice. In this study, we investigated the mechanisms of action of MRA in a rat model of limbal deficiency.Methods: CN was induced in Lewis rats by total corneal de‐epithelialization and limbal scratching. Spironolactone (SPL, 25 mg/kg) or eplerenone was administered daily. Control eyes received vehicle. Rats were euthanized at day 3 and 7, and corneas were dissected for transcriptomic analysis. Proteins extracted from corneas at day 7 were used for western blot. Corneal innervation was evaluated by immunostaining of TUBB3 on corneal flat mounts at day 16. Corneal morphology and CN were assessed in vivo by Micron III optical coherence tomography and fluorescein angiography at day 14.Results: MR antagonists (MRA) favoured epithelial regeneration and nerve growth, reduced corneal angiogenesis, edema and inflammation in a rat model of CN. Transcriptomic analysis showed 75 differentially expressed genes in SPL groups. Gene set enrichment analysis highlighted pathways including wound healing and differentiation, inflammatory and immune responses, hypoxia, angiogenesis and neuroprotection. SPL upregulated Dcn, Col1a1, Timp2 and downregulated Lrg1 at day 3, and induced Pedf at day 7. Increase in DCN protein by SPL was confirmed by western blot.Conclusions: MRA regulates genes/proteins involved in anti‐inflammatory and anti‐angiogenic processes, and improves corneal re‐innervation in CN model. Dcn is one of the downstream regulated targets.

A Coculture System for Modeling Intestinal Epithelial-Fibroblast Crosstalk.

Epithelial organoid monoculture is a powerful tool to model stem cell dynamics in vitro. However, extensive efforts have recently revealed various niche players and their significant roles in regulating epithelial stem cells. Among these niche components, fibroblasts have been heavily recognized in the field as a critical niche signal secretor. Thus, understanding the roles of fibroblasts in epithelial dynamics has become increasingly relevant and crucial. This propels the development of approaches to coculture epithelial 3D organoids with fibroblasts to model epithelial-fibroblast crosstalk in vitro. Here, we describe a stepwise coculture method to isolate and culture primary intestinal fibroblasts and epithelial organoids together. Aligned with the recent literature, our coculture protocol allows for primary intestinal fibroblast support of epithelial organoid growth.

Endoscopic-Assisted Excision of an Extensive Inverted Papilloma (Krouse Stage IV) through Lateral Rhinotomy Approach: A Case Report

Inverted papilloma is a benign epithelial growth arising from the Schneiderian mucosa with a tendency to grow into the underlying stroma. The etiology remains obscure but its tendency for an association with malignancy makes it an important entity important for the surgeon. We present a case report of an extensive inverted papilloma (Krouse stage IV). The tumour involved entire right side nasal cavity extending to the vestibule, extended to the orbit and also intracranially (though extradural) through the frontal and ethmoid sinuses. This extensive tumour was removed by a combined (lateral rhinotomy plus endoscopic) approach and obviated the need for external approach to frontal sinus as well as extended lateral rhinotomy. Thus, even for extensive tumours using the endoscope as an adjunct aids in complete tumour removal and prevents additional morbidity to the patient.

Combining EGFR and KRAS G12C Inhibitors for KRAS G12C Mutated Advanced Colorectal Cancer

KRAS is a commonly mutated gene in advanced colorectal cancer (CRC). Recently, inhibitors of KRAS G12C were developed and have shown promising efficacy for KRAS G12C mutated non-small cell lung cancer. However, KRAS G12C inhibitor monotherapy has not demonstrated excellent efficacy for KRAS G12C mutated advanced CRC due to multiple resistance mechanisms, especially receptor tyrosine kinase (RTK) signaling activation. To overcome this resistance mechanism, various combinations of epithelial growth factor receptor (EGFR) and KRAS G12C inhibitors, including panitumumab plus sotorasib, have been investigated in clinical trials. The combination of EGFR and KRAS G12C inhibitors for KRAS G12C mutated CRC demonstrated overall response rates ranging from 26% to 62.5% in seven clinical trials of phase I to III, whose data are available so far. The median progression-free survival in these trials ranged from 3.9 to 8.1 months. These efficacy data suggest that KRAS G12C inhibitor combination with EGFR inhibitors is more effective for KRAS G12C mutated advanced CRC than KRAS G12C inhibitor monotherapy. They also showed reasonable safety of the combination regimen. Based on these results, phase III clinical trials are being conducted to investigate EGFR and KRAS G12C inhibitor combinations as a first or second-line treatment for KRAS G12C mutated advanced CRC. Furthermore, other KRAS G12C inhibitors, KRAS G12D inhibitors, and pan-RAS inhibitors are being developed, which could make more patients with advanced CRC eligible for KRAS inhibition.