Uniform Expression Research Articles

Clinical Significance of FAM83G in Breast Cancer: Association With Triple-negative Subtype and Prognosis.

Family with sequence similarity 83 member G (FAM83G) plays an important role in cancer aggressiveness and patients' prognosis across various types of cancer. However, the association of FAM83G protein expression in breast cancer with clinicopathologic factors, breast cancer subtypes, and prognosis is not well characterized. This study aimed to elucidate the clinical significance of FAM83G protein expression in breast cancer. Immunohistochemistry was employed to examine FAM83G protein expression in 75 breast cancer tissues, assessing its potential as a biomarker for breast cancer patients. The Kaplan-Meier plotter was used to estimate the correlation between FAM83G mRNA expression and survival outcomes in TNBC patients. FAM83G protein was predominantly localized in the cytoplasm of breast cancer cells, exhibiting uniform expression throughout tumor tissues. FAM83G expression was significantly higher in cancerous areas compared to non-cancerous areas. High FAM83G expression was more prevalent in triple-negative breast cancer (TNBC) cases than in hormone receptor-positive cases. Although high FAM83G protein expression did not significantly impact prognosis in our cohort, a large-scale database analysis revealed an association between high FAM83G expression and poor prognosis in TNBC cases. This study demonstrates an association between high FAM83G expression in breast cancer tissues and TNBC. FAM83G may serve as a promising biomarker and therapeutic target for breast cancer patients.

Expression of soluble guanylate cyclase (sGC) and its ability to form a functional heterodimer are crucial for reviving the NO-sGC signaling in PAH

In order to determine the underpinnings of a dysfunctional NO-sGC signal pathway which occurs in pulmonary arterial hypertension (PAH), we investigated pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients. We found low expression of sGC, a poor sGCα1β1 heterodimer and this correlated with low expression of its facilitator chaperon, hsp90. Treating PASMCs overnight (16 h) with low micromolar doses of a slow release NO donor DETANONOate, reinstated the sGCα1β1 heterodimer and restored its NO-heme dependent activity. Transwell co-culture of HEK cells stably expressing eNOS with PAH PASMCs also restored the sGC heterodimer and its heme-dependent activity with sGC stimulator, BAY 41–2272. To determine whether the dysfunctionality in the NO-sGC pathway stems from a dysfunctional eNOS producing negligible NO, we did transwell co-cultures of pulmonary arterial endothelial cells (PAECs) with PASMCs. Our results indicated that PAECs from both control and PAH samples when activated for eNOS restored both sGC heterodimer and its heme-dependent sGC activity in the corresponding PASMCs, suggesting that PAECs from PAH can also generate NO. In line with these results expression of eNOS, its support chaperon hsp90, its specific kinase Akt, p-Akt or post-translational modifications (PTMs) like OGlcNAc or phospho-tyrosine were unchanged in PAH relative to controls. Additionally there was uniform expression of Hbα/β and Mb in PASMCs or PAECs in PAH or controls and these globins can effectively scavenge the eNOS generated NO, as there was evidence of strong eNOS-Hb/Mb interactions. Our studies suggest that factors such as globin NO scavenging along with vascular remodeling in PAH can cause hampered vasodilation which in the face of poor NO levels as occurs in PAH are additional impediments for effective vasodilation. However importantly our studies suggests that future therapies can use low doses of NO along with sGC stimulators as a potential drug for PAH subjects.

A robust synthetic biology toolkit to advance carboxysome study and redesign.

Carboxysomes are polyhedral protein organelles that microorganisms use to facilitate carbon dioxide assimilation. They are composed of a modular protein shell which envelops an enzymatic core mainly comprised of physically coupled Rubisco and carbonic anhydrase. While the modular construction principles of carboxysomes make them attractive targets as customizable metabolic platforms, their size and complexity can be a hinderance. In this work, we design and validate a plasmid set - the pXpressome toolkit -in which α-carboxysomes are robustly expressed and remain intact and functional after purification. We tested this toolkit by introducing mutations which influence carboxysome structure and performance. We find that deletion of vertex-capping genes results in formation of larger carboxysomes while deletion of facet forming genes produces smaller particles, suggesting that adjusting the ratio of these proteins can rationally affect morphology. Through a series of fluorescently labeled constructs, we observe this toolkit leads to more uniform expression and better cell health than previously published carboxysome expression systems. Overall, the pXpressome toolkit facilitates the study and redesign of carboxysomes with robust performance and improved phenotype uniformity. The pXpressome toolkit will support efforts to remodel carboxysomes for enhanced carbon fixation or serve as a platform for other nanoencapsulation goals.

Detection of Residual iPSCs Following Differentiation of iPSC-Derived Retinal Pigment Epithelial Cells.

Purpose: The goal of this study was to develop a lot release assay for iPSC residuals following directed differentiation of iPSCs to retinal pigment epithelial (RPE) cells. Methods: RNA Sequencing (RNA Seq) of iPSCs and RPE derived from them was used to identify pluripotency markers downregulated in RPE cells. Quantitative real time PCR (qPCR) was then applied to assess iPSC residuals in iPSC-derived RPE. The limit of detection (LOD) of the assay was determined by performing spike-in assays with known quantities of iPSCs serially diluted into an RPE suspension. Results: ZSCAN10 and LIN28A were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs ZSCAN10 and LIN28A were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of ZSCAN10 and LIN28A in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, ZSCAN10, and LIN28A expression in iPSCs was generally uniform. The LOD for ZSCAN10 and LIN28A in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of ΔΔCt found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC/10,000 RPE cells in both iPSC lines. Conclusions: qPCR for ZSCAN10 and LIN28A detects <1 in 10,000 residual iPSCs in a population of iPSC-derived RPE providing an adequate LOD of iPSC residuals for lot release testing.

Study on the scattering characteristics of large double-spherical systems

To study the influence of multiple scattering factors on the scattering characteristics, the scattering properties of a sizable double-spherical system at random orientations were analyzed by comparison of the T matrix and simple addition in the S-, C-, and X-bands, including the backscatter section (Cbak), attenuation section (Cext) and scattering section (Csca), asymmetry factor (g), and single scattering albedo (w), with the change in the spherical distance (d) and particle radius (r). It was found that the spacing between the spheres is four times the sphere’s radius, which is a clear cut-off point for the influence of multiple scattering in a two-spherical system. Then, by fitting the relative difference multiples of the T matrix method and the simple addition method, the uniform expression of each optical parameter under multiple scattering conditions is obtained.

The Role of Claudins in the Pathogenesis of Dextran Sulfate Sodium-Induced Experimental Colitis: The Effects of Nobiletin.

The pathogenesis of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease is not well understood. This study investigated the roles and regulation of the claudin-1, -2, -3, and -4 isoforms in the pathogenesis of ulcerative colitis, and the potential therapeutic effects of nobiletin. Colitis was induced in rats by administering dextran sulfate sodium [DSS] in drinking water for seven days. Animals were treated daily with nobiletin [oral, 60 mg/Kg body weight] and studied in four groups, C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. On day seven, the animals were sacrificed, and colonic tissues were collected and analyzed. Both macroscopic and microscopic findings suggest the progression of colitis. In the inflamed colon, claudin-1 and -4 proteins were decreased, claudin-2 increased, while the claudin-3 protein remained unchanged. Except for claudin-1, these changes were not paralleled by mRNA expression, indicating a complex regulatory mechanism. Uniform β-actin expression along with consistent quality and yield of total RNA indicated selectivity of these changes. Nobiletin treatment reversed these changes. Altered expression of the claudin isoforms -1, -2, and -4 disrupts tight junctions, exposing the lamina propria to microflora, leading to electrolyte disturbance and the development of ulcerative colitis. Nobiletin with its anti-inflammatory properties may be useful in IBD.

A Knitted and MXenzyme-Integrated Dressing for Geriatrics Diagnosis and Ulcer Healing.

Integrated diagnostic and therapeutic dressings are desirable to relieve diabetic patients who often suffer from diabetic foot ulcers (DFUs) and peripheral vascular diseases (PVDs). However, it is highly difficult to monitor the pulse waves with fidelity under wet environments and connect the waveforms to diseases through a small strain sensor. Additionally, immobilizing MXenzyme to regulate spatially heterogeneous levels of reactive oxygen species (ROS) and applying active intervention to enhance ulcer healing on a single structure remain a complex task. To address these issues, we designed a multiscale wearable dressing comprising a knitted all-textile sensing array for quantitatively investigating the pulse wave toward PVD diagnosis. MXenzyme was loaded onto the dressing to provide multiple enzyme mimics for anti-inflammatory activities and deliver electrical stimulation to promote wound growth. In mice, we demonstrate that high and uniform expression of the vascular endothelial growth factor (VEGF) is observed only in the group undergoing dual mediation with electrical stimulation and MXenzyme. This observation indicates that the engineered wound dressing has the capability to accelerate healing in DFU. In human patient evaluations, the engineered dressing distinguishes vascular compliance and pulse period, enabling the diagnosis of arteriosclerosis and return blockage, two typical PVDs. The designed and engineered multiscale dressing achieves the purpose of integrating diagnostic peripheral vessel health monitoring and ulcer healing therapeutics for satisfying the practical clinical requirements of geriatric patients.

Abstract Mo058: Cellular and Subcellular Ventricular Localization of the Fast Transient Outward Potassium Channels in the Murine Heart

Introduction: Cardiac fast transient outward potassium currents (I to,f ) are formed by KCND2 (Kv4.2) and KCND3 (Kv4.3) channels in mice and primarily KCND3 in humans. Rapid I to,f activation induces early phase 1 repolarization of action potentials (APs), which has been linked strongly to the modulation of excitation-contraction coupling (ECC). Consequently, variations in I to,f densities underlie regional differences in ventricular APs and myocardial contractile properties. To investigate further the role of I to,f in cardiac function, we created mice with fluorescently labelled KCND2 (KCND2-GFP) and KCND3 (KCND3-RFP). Methods and Results: The expression patterns of I to,f in the ventricles were examined using frozen histological sections. Remarkably, KCND2 and KCND3 were preferentially localized to the intercalated discs (ICDs) by &gt;50%, with expression also in lateral membranes and within t-tubules (i.e. a sarcomeric pattern), yet KCND3 showed a greater t-tubule localization. Additionally, KCND2 expression was, as expected, greater ( P &lt;0.048, two-way ANOVA; n=3) in the epi- versus endo-myocardium of both ventricles, whereas KCND3 lacked ( P &gt;0.172; n=3) a distinct gradient. Moreover, KCND2 expression inversely correlated with the temporal sequence of ventricular activation, whereby the posterobasal regions that depolarize last show elevated Kv4.2 ( P =0.009, unpaired t-test; n=3) compared to the anterolateral endocardium activated first. In contrast, Kv4.3 demonstrates a relatively uniform expression. Conclusions: The regional expression patterns of I to,f in ventricles and preferential I to,f expression in t-tubules support previous studies suggesting that I to,f controls the regional variations in the timing and amplitude of ECC. We are currently exploring the possible differential actions of KCND2 versus KCND3 in ECC and the potential consequence of ICD localization of I to,f on ephaptic coupling.

Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells.

Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB. We performed single-cell RNA sequencing on six normal placentae, four from early (6-8 weeks) and two from late (12-14 weeks) first trimester, of which two of the early first trimester cases were separated into basal (maternal) and chorionic (fetal) fractions prior to sequencing. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC. CTB clusters displayed notable distinctions based on gestational age, with early first trimester placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched in early first trimester placentae, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5-CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage.

A unified explicit charge-based capacitance model for metal oxide thin-film transistors

A unified and complete capacitance model of metal oxide thin-film transistors (MO TFTs) based on three-terminal charges is proposed in this paper. The analytical expression of the three-terminal charges is obtained with the effective charge density approach and the Ward-Dutton charge partitioning approach. By considering the non-reciprocal capacitance between any two terminals, the complete capacitance model of the MO TFTs is proposed with an accurate description. The proposed model has a uniform and analytical capacitance expression over the full working regions with a specific physical meaning based on the surface potential solution. Furthermore, the sufficient capacitance experimental data of the fabricated IZO-TFT are presented to verify the proposed model. It is shown that there is a good agreement between the experimental data and the proposed model in a wide range of working regions.

Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

BackgroundIn the treatment of oral cavity cancer, margin status is one of the most critical prognostic factors. Positive margins are associated with higher local recurrence and lower survival rates. Therefore, the universal goal of oral surgical oncology is to achieve microscopically clear margins. Near-infrared fluorescence guided surgery (FGS) could improve surgical resection using fluorescent probes. αVβ6 integrin has shown great potential for cancer targeting due to its overexpression in oral cancers. Red fluorescent contrast agent IRDye 680 coupled with anti-αVβ6 peptide (IRDye-A20) represents an asset to improve FGS of oral cancer. This study investigates the potential of IRDye-A20 as a selective imaging agent in 3D three-dimensional tongue cancer cells.MethodsαVβ6 integrin expression was evaluated by RT-qPCR and Western Blotting in 2D HSC-3 human tongue cancer cells and MRC-5 human fibroblasts. Targeting ability of IRDye-A20 was studied in both cell lines by flow cytometry technique. 3D tumor spheroid models, homotypic (HSC-3) and stroma-enriched heterotypic (HSC-3/MRC-5) spheroids were produced by liquid overlay procedure and further characterized using (immuno)histological and fluorescence-based techniques. IRDye-A20 selectivity was evaluated in each type of spheroids and each cell population.ResultsαVβ6 integrin was overexpressed in 2D HSC-3 cancer cells but not in MRC-5 fibroblasts and consistently, only HSC-3 were labelled with IRDye-A20. Round shaped spheroids with an average diameter of 400 μm were produced with a final ratio of 55%/45% between HSC-3 and MRC-5 cells, respectively. Immunofluorescence experiments demonstrated an uniform expression of αVβ6 integrin in homotypic spheroid, while its expression was restricted to cancer cells only in heterotypic spheroid. In stroma-enriched 3D model, Cytokeratin 19 and E-cadherin were expressed only by cancer cells while vimentin and fibronectin were expressed by fibroblasts. Using flow cytometry, we demonstrated that IRDye-A20 labeled the whole homotypic spheroid, while in the heterotypic model all cancer cells were highly fluorescent, with a negligible fluorescence in fibroblasts.ConclusionsThe present study demonstrated an efficient selective targeting of A20FMDV2-conjugated IRDye 680 in 3D tongue cancer cells stroma-enriched spheroids. Thus, IRDye-A20 could be a promising candidate for the future development of the fluorescence-guided surgery of oral cancers.Graphical

Psychological Well-being and the Structure of the teacher’s Agency

Introduction. In positive psychology, psychological well-being is associated with personal resources that ensure creative processes and positive changes in a person’s life and activities. Considering agency as the main professional and personal resource of a teacher for positive transformations in modern education, we assume that agency acts as such a resource provided that the teacher perceives himself as a psychologically prosperous person. Psychological well-being in the study is considered within the framework of the PERMA model and represents the teacher’s self-representation of the main resources of positive functioning (positive emotions, involvement in relationship activities, meaning and achievement in activities) and his experience of happiness and life satisfaction. The purpose of the study is to study the characteristics of the psychological well-being of a modern teacher and the relationship between the components of well-being and the structure of the teacher’s agency.Materials and Methods. The severity of the components of teachers’ subjectivity was studied using the “Structure of agency” questionnaire (E. N. Volkova, I. A. Seregina). To study psychological wellbeing, the Russian version of the PERMA-Profiler questionnaire was used (O. M. Isaeva, A. Yu. Akimova, E. N. Volkova). 447 people participated in the study, of which 376 people. (84.1 %) – women, 71 people. (15.9 %) – men. The average age of study participants was 37.2 years (standard deviation SD=12.5). Work experience – from 1 year to 47 years.Results. The results of the study showed that the general level of well-being, as well as the values of the main components of well-being among modern teachers, is in the range of average values, however, all components of psychological well-being are expressed among teachers to a lesser extent than among representatives of other professional groups and in general among respondents of the Russian sample. Stratification variables that influence the psychological well-being of school teachers are the characteristics of age, work experience, and qualification category. The psychological wellbeing of teachers corresponds to older age, decreased work experience, and a low qualification category (or lack thereof). Psychologically well-off teachers are characterized by a balanced structure of agency, which presupposes uniform and high expression of all its components. Among the components of agency, psychological well-being is most determined by the teacher’s ability to make personal choice and the development of reflection.Discussion and Conclusions. The teacher’s psychological well-being can be considered as a characteristic of his/her professional socialization connected with the level of students' well-being. The actual level of teacher’s psychological well-being is lower than in the general Russian sample and in the sample of other professional pedagogical groups - preschool teachers. The low level of the teacher's psychological well-being can hinder the teacher's professional development. The teacher's psychological well-being is correlated with the teacher’s agency, in particular, with the teacher's activity, choices and responsibility, reflection. A balanced structure of agency is correlated the psychological well-being of teachers and the understanding another person is expressed to a significant extent.

Steady-state, therapeutic, and helminth-induced IL-4 compromise protective CD8 T cell bystander activation.

Memory CD8 T cells (Tmem) can be activated into innate-like killers by cytokines like IL-12, IL-15, and/or IL-18; but mechanisms regulating this phenomenon (termed bystander activation) are not fully resolved. We found strain-intrinsic deficiencies in bystander activation using specific pathogen-free mice, whereby basal IL-4 signals antagonize IL-18 sensing. We show that therapeutic and helminth-induced IL-4 impairs protective bystander-mediated responses against pathogens. However, this IL-4/IL-18 axis does not completely abolish bystander activation but rather tunes the expression of direct versus indirect mediators of cytotoxicity (granzymes and interferon-γ, respectively). We show that antigen-experience overrides strain-specific deficiencies in bystander activation, leading to uniform IL-18 receptor expression and enhanced capacity for bystander activation/cytotoxicity. Our data highlight that bystander activation is not a binary process but tuned/deregulated by other cytokines that are elevated by contemporaneous infections. Further, our findings underscore the importance of antigen-experienced Tmem to dissect the contributions of bystander Tmem in health and disease.

Neuroectodermal influence in odontogenic cyst and tumor: evidence through CD56 immunoexpression.

CD56, associated with neuroectodermal differentiation of the embryonal cells, is often considered a marker of neural lineage. Odontogenic keratocysts (OKCs) are of particular interest because of their characteristic histopathologic features, high recurrence rate, and aggressive behavior. CD56 immunoreactivity in these lesions has been reported with very high frequency. The present study analyzes the immunoexpression of CD56 in ameloblastoma (AM) and OKC to infer neuroectodermal influence in the pathogenesis of odontogenic lesions and its correlation with clinicopathologic parameters. Fifty histopathologically confirmed cases of OKC and AM, 25 from tooth-bearing (TB) and molar-ramus (MR) regions each, and 5 dental follicular tissues as control were collected from the department archives and immunohistochemical analysis with CD56 was carried out. CD56 immunopositivity was seen in 64% AM and 36% OKC cases. The majority of AM cases showed cytoplasmic expression in the peripheral cells of odontogenic islands; similarly, OKC cases showed continuous and uniform cytoplasmic expression in the basal and parabasal cells of the cystic lining. CD56 immunopositivity was found in more AM cases as compared to OKC cases in both the TB and MR regions. The assessment of CD56 immunoexpression in odontogenic cyst and tumor (AM) may aid in understanding the role of neuroectodermal influence in the etiopathogenetic pathways and a possible influence of CD56 on the clinical behavior and aggressiveness of the odontogenic lesions. A correlation of CD56 expression with the clinical outcome of the disease (site, perforation, root resorption, and tooth displacement) can help envisage possible prognostic assessment for these lesions.

Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

Diagnostic Value of Immunostaining for Thyroid Transcription Factor 1 (TTF1) and Paired Box 8 (PAX8) in Distinguishing Pulmonary Metastases of Mesonephric and Mesonephric-like Adenocarcinomas from Primary Lung Adenocarcinomas.

Both mesonephric adenocarcinoma (MA) and mesonephric-like adenocarcinoma (MLA) express thyroid transcription factor 1 (TTF1). TTF1 is also considered a highly sensitive and specific diagnostic marker for primary lung adenocarcinoma (PLA). However, distinguishing PLA from pulmonary metastatic MA/MLA (PMM) based on the expression of TTF1 alone can be difficult. This study aimed to investigate the expression of TTF1 and paired box 8 (PAX8) and assess their value in distinguishing PMM from PLA. We reviewed the electronic medical records and pathology slides of eight PMM cases. We conducted immunostaining for TTF1 and PAX8 in 6, 8, and 21 cases of primary MA/MLA, PMM, and PLA, respectively. Two patients with stage IB uterine MLA developed lung metastases at 5 and 57 months after hysterectomy. Solitary pulmonary nodules were suspected to be primary lung cancer in two patients. Compared to primary tumors, all matched PMMs exhibited reduced TTF1 immunoreactivity. In contrast, the majority of PLAs showed uniform and intense TTF1 expression. All except one PMM exhibited diffuse and strong PAX8 expression, while only one PLA showed focal and weak PAX8 expression. Immunostaining for TTF1 and PAX8 can help in distinguishing PMM from PLA in the diagnosis of pulmonary lesions detected in patients with a history of MA/MLA.

Combined spatial Keratin expression profiles at the invasive front represent a prognostic classifier for head and neck cancer

Keratin proteins are intermediate filaments that provide mechanical support and guide a plethora of important functions in epithelial cells. Although Keratins can be effectively used to characterize and differentiate cell lineages in many epithelia, it is still unclear if combinations of Keratin types and their spatial expression patterns can aid prognostication of head and neck squamous cell carcinoma (HNSCC). Here, we have assessed a panel of Keratin markers in four HPV negative HNSCC patient-derived organoid (PDO) models and their corresponding primary patient tissue to define spatial expression profiles of basal and supra-basal Keratins during invasion in Collagen-I matrices. Based on these profiles we have devised a scoring classification consisting of the following profiles: (i) no expression (Negative), (ii) uniform expression (Homogenous), (iii) heterogeneous expression (Mosaic), (iv) expression in the tumor core (Core), and (v) expression at the invasive front (Edge). Subsequently, this 5-tier system was validated in a well-documented tissue cohort of 157 HNSCC patients. We observe that patients with a Keratin 13 (K13) Edge profile have a significantly better prognosis than patients with K13 Core expression or K13 Negative tumors. Interestingly, we also find that K14 mosaicism strongly correlates with a favorable prognosis. We show that the absence of K13 from the tumor edge, in combination with K14 homogenous expression is a strong biomarker for poor prognosis in HNSCC. In short, our work indicates that defining the spatial expression patterns of basal and supra-basal markers in invasive HNSCC can benefit patient prognostication.

ℏ2 Corrections to Semiclassical Transmission Coefficients.

The uniform semiclassical expression for the energy-dependent transmission probability through a barrier has been a staple of reaction rate theory for almost 90 years. Yet, when using the classical Euclidean action, the transmission probability is identical to 1/2 when the energy equals the barrier height since the Euclidean action vanishes at this energy. This result is generally incorrect. It also leads to an inaccurate estimate of the leading order term in an ℏ2n expansion of the thermal transmission coefficient. The central result of this paper is that adding an ℏ2 dependent correction to the uniform semiclassical expression, whether as a constant action or as a shift in the energy scale, not only corrects this inaccuracy but also leads to a theory that is more accurate than the previous one for almost any energy. Shifting the energy scale is a generalization of the vibrational perturbation theory 2 (VPT2) and is much more accurate than the "standard" VPT2 theory, especially when the potential is asymmetric. Shifting the action by a constant is a generalization of a result obtained by Yasumori and Fueki (YF) only for the Eckart barrier. The resulting modified VPT2 and YF semiclassical theories are applied to the symmetric and asymmetric Eckart barrier, a Gaussian barrier, and a tanh barrier. The one-dimensional theories are also generalized to many-dimensional systems. Their effect on the thermal instanton theory is discussed.

Abstract LB402: In vivo efficacy of anti-B7-H3 antibody-drug conjugate (ADC) ifinatamab deruxtecan (I-DXd; DS-7300): An update from the Pediatric Preclinical In Vivo Testing (PIVOT) Program

Abstract Introduction: I-DXd (DS-7300) is a B7-H3-directed ADC with a topoisomerase 1 inhibitor payload (an exatecan derivative, DXd). I-DXd has demonstrated clinical efficacy in adults with heavily pretreated solid tumors (Patel et al., ESMO 2023). We previously reported that I-DXd induced objective responses in PDX models for multiple B7-H3 (CD276) expressing pediatric histologies, including rhabdomyosarcoma, osteosarcoma, neuroblastoma, and Wilms tumor. We extend these results with dose-response testing and include an isotype control ADC (IC-ADC) to evaluate whether B7-H3 expression is required for optimal response. Methods: PDX models were dosed with vehicle, I-DXd, or IC-ADC, the latter two at 1, 3, and 10 mg/kg intravenously on days 1 and 15. Testing was performed using 5 animals per treatment group. Activity was assessed by the PIVOT objective response measure (ORM) (Ped Blood Cancer 2007;49:928-940) that defines objective response as partial, complete, or maintained complete response (PR, CR, and MCR) compared to stable disease (SD) or progressive disease, with or without growth delay (PD2 and PD1, respectively). Results: I-DXd showed dose-dependent activity with MCR for all 5 models at 10 mg/kg. PR or MCR responses were observed in 4 of 5 models to I-DXd at 3 mg/kg, with no objective responses to I-DXd at 1 mg/kg. Only the RMS model showed objective response to IC-ADC. For all models I-DXd was significantly more active than IC-ADC. Conclusions: We confirm significant anti-tumor activity for I-DXd for multiple pediatric solid tumors with B7-H3 expression and provide evidence of a dose response effect for I-DXd and for enhanced efficacy for I-DXd versus IC-ADC. Testing in additional preclinical models (neuroblastoma and glioblastoma) is underway. Due to the uniform high expression of B7-H3 in multiple pediatric histologies, I-DXd warrants evaluation for clinical activity in pediatric solid tumor patients. TABLE 1. NAND Activity for DS-7300a and Isotype Control using the PIVOT Objective Response Measure (ORM) Line IHC B7-H3 H-Score Diagnosis DS-7300a Isotype Control 1 mg/kg 3 mg/kg 10 mg/kg 1 mg/kg 3 mg/kg 10 mg/kg OS-9 230 Osteosarcoma PD2 PR MCR PD1 PD1 PD1 OS-33 240 Osteosarcoma PD2 MCR MCR PD1 PD2 SD Rh30 140 Rhabdomyosarcoma PD2 MCR MCR PD1 PR MCR WT11 150 Wilms tumor PD1 PD2 MCR PD1 PD1 PD1 Felix 210 Neuroblastoma PD2 MCR MCR PD1 PD1 PD2 Citation Format: Richard Gorlick, Jonathan Gill, Wendong Zhang, Yael Mosse, John Maris, David Groff, Malcolm Smith, Beverly Teicher, Steve Neuhauser, Tim Stearns, Vivek Philip, Emily Jocoy, Jeff Chuang, Carol J. Bult, Jun Hasegawa, Xiaozhong Qian, Peter Houghton, Raushan Kurmasheva. In vivo efficacy of anti-B7-H3 antibody-drug conjugate (ADC) ifinatamab deruxtecan (I-DXd; DS-7300): An update from the Pediatric Preclinical In Vivo Testing (PIVOT) Program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB402.

Induction of tauopathy in a mouse model of amyloidosis using intravenous administration of adeno-associated virus vectors expressing human P301L tau.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which extracellular aggregates of the amyloid beta (Aβ) peptide precede widespread intracellular inclusions of the microtubule-associated protein tau. The autosomal dominant form of AD requires mutations that increase production or aggregation of the Aβ peptide. This has led to the hypothesis that amyloid deposition initiates downstream responses that lead to the hyperphosphorylation and aggregation of tau. Here we use a novel approach, somatic gene transfer via intravenous adeno-associated virus (AAV), to further explore the effects of pre-existing amyloid deposits on tauopathy. APP+PS1 mice, which develop amyloid deposits at 3 to 6 months of age, and non-transgenic littermates were injected at 8 months of age intravenously with AAV-PHP.eB encoding P301L human tau. Tissue was collected at 13 months and tauopathy was assessed. Total human tau expression was observed to be relatively uniform throughout the brain, reflecting the vascular route of AAV administration. Phospho-tau deposition was not equal across brain regions and significantly increased in APP+PS1 mice compared to non-transgenic controls. Interestingly, the rank order of phospho-tau deposition of affected brain regions in both genotypes paralleled the rank order of amyloid plaque deposits in APP+PS1 mice. We also observed significantly increased MAPT RNA expression in APP+PS1 mice compared to non-transgenic despite equal AAV transduction efficiency between groups. This model has advantages over prior approaches with widespread uniform human tau expression throughout the brain and the ability to specify the stage of amyloidosis when the tau pathology is initiated. These data add further support to the amyloid cascade hypothesis and suggest RNA metabolism as a potential mechanism for amyloid-induced tauopathy.