Epithelial Growth Research Articles

AQP3-Dependent PI3K/Akt Modulation in Breast Cancer Cells.

Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated H2O2 uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2-related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.

A pathogenic role for regulatory T cells in chronic liver disease

Abstract Current dogma holds that regulatory T (Treg) cells preserve tissue function in settings of inflammation and damage through their anti-inflammatory activity and production of epithelial growth factors – the latter of which is independent from immunosuppression. Consistent with this, we observe that Treg cells – in particular those producing the epidermal growth factor receptor (EGFR)-ligand amphiregulin (Areg) – are enriched in the livers of mice and humans with non-alcoholic steatohepatitis (NASH). Mouse and human liver Treg cells undergo substantial transcriptional changes in chronic liver damage, reflecting their increased activation; however, rather than playing a protective role, we find that Treg cell–derived Areg promotes NASH-induced liver fibrosis through the direct activation of EGFR on hepatic stellate cells. Clinically, NASH is closely linked to insulin resistance, but the mechanistic contributions of NASH-induced disease processes to insulin resistance remain unclear. We further observe that Treg cell–derived Areg promotes glucose intolerance in a NASH-dependent manner – through the induction of hepatic gluconeogenesis – a process also mediated through EGFR signaling on hepatic stellate cells. Mechanistically, Treg cell–derived Areg promotes hepatocyte gluconeogenesis through hepatocyte detection of soluble mediators produced by Areg-activated hepatic stellate cells – offering new insight into how chronic liver disease promotes insulin resistance. Taken together, we provide the first evidence that Treg cells mediate a maladaptive role in tissue injury, finding that their production of a growth factor plays a central role in liver disease and promotes liver fibrosis and glucose intolerance in NASH. This research was supported by grants from NIH (K22 AI127847, R01 HL148718, R01 CA259634, T32 GM007367, P30 CA013696, P30 DK132710) and the Searle Scholars Program (SSP-2017-2179).

A rare case of tertiary nasal syphilis: an unusual ear, nose, and throat manifestation of a rare disease

Tertiary syphilis is a rare entity in present antibiotic era. Syphilis of the nose is considered to be very rare by all authorities. It is mainly a systemic disease caused by the spirochete Treponema pallidum. The infection acquired may be congenital or acquired. Tertiary syphilis shows most marked manifestations in nose, causing superficial and deep ulcerations, and gumma. Nasal septum, especially the junction of the cartilaginous with the bony septum resulting in perforation is a usual manifestation. In cases where the bony septum is involved, existence of syphilis is unquestionable. The nasal deformity results from destruction of bony framework of nose and shrinking of fibroid tissue, thus, produces the typical saddle nose which is characteristic of syphilis. The treatment line for syphilis has remained relatively unchanged in recent years and continues to vary with stage of infection. However, local treatment consists of clearance of crusts and regular cleansing of the nasal passages by copious alkaline douches. Yellow mercury oxide ointment may be applied locally. The purpose of local treatment is to remove the discharge and crusts, kill spirochetes, promote wound healing and epithelial growth, prevent secondary infection.

Neurodifferentiation and Neuroprotection Potential of Mesenchymal Stromal Cell-Derived Secretome Produced in Different Dynamic Systems.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein (α-synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrow-derived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α-synuclein in one Caenorhabditis elegans model of PD. Results showed that secretomes from both systems were able to induce neurodifferentiation, though the secretome produced in the SP system had a greater effect. Additionally, in the conditions of our study, only the secretome produced in SP had a neuroprotective potential. Lastly, the secretomes had different profiles regarding the presence and/or specific intensity of different molecules, namely, interleukin (IL)-6, IL-4, matrix metalloproteinase-2 (MMP2), and 3 (MMP3), tumor necrosis factor-beta (TNF-β), osteopontin, nerve growth factor beta (NGFβ), granulocyte colony-stimulating factor (GCSF), heparin-binding (HB) epithelial growth factor (EGF)-like growth factor (HB-EGF), and IL-13. Overall, our results suggest that the culture conditions might have influenced the secretory profiles of cultured cells and, consequently, the observed effects. Additional studies should further explore the effects that different culture systems have on the secretome potential of PD.

Engineered synthetic nanobody-based biosensors for electrochemical detection of epidermal growth factor receptor

Two engineered synthetic nanobody-based nanobiocomposite platforms were developed for label-free electrochemical detection of the epithelial growth factor receptor (EGFR) biomarker. Screen-printed carbon electrodes (SPCE) were decorated either with NiO nanoparticles (NPs) or poly(thiophene acetic acid) (PTAA) to link the anti-EGFR nanobody (Nb) and form nanobiocomposites for detecting the EGFR biomarker by electrochemical impedance spectroscopy (EIS). The nanoarchitectures were prepared by in situ electrosynthesis of NiO NPs or PTAA layers at SPCEs. A modified version of the 9G8 Nb (Nb9G8m), specific for the EGFR (anti-EGFR), was designed and produced as the nanobiosensor bioreceptor. This Nb was engineered to provide a hexahistidine tag (6xHis-tag) and a lysine (Lys) dual functionality to form a (6xHis-tag)/Ni2+ or Lys/PTAA interface. The biosensing interfaces were characterized by field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, cyclic voltammetry, and EIS. The nanobody/nanobiocomposite-based biosensors detected EGFR proteins in a linear range from 0.25 to 50 μg mL−1 and 0.5 to 50 μg mL−1, with limits of detection of 0.46 μg mL−1 and 1.14 μg mL−1, for NiO- and PTAA-based platforms, respectively. The biosensing platforms offer high simplicity, specificity, and selectivity to detect EGFR, but Nbs can be readily engineered to detect other (glycol)proteins. Finally, as a proof of concept, the EGFR was detected in several tumor cell lines, differentiating biomarker expression among them.

Prepartum Magnesium Butyrate Supplementation of Dairy Cows Improves Colostrum Yield, Calving Ease, Fertility, Early Lactation Performance and Neonatal Vitality.

Butyrate promotes rumen epithelium growth and function; however, the effect of prepartum butyrate supplementation on dairy cow productivity, health and their offspring has not been extensively studied. Furthermore, no studies have investigated the effect of magnesium butyrate (MgB), which is also a source of magnesium. A trial was performed to test the hypothesis that prepartum MgB supplementation (105 g/cow/day) would increase colostrum quality and improve calving, newborn calf vitality and cow health. Multiparous Holstein cows were randomly assigned to MgB supplemented (n = 107) and Control groups (n = 112). Colostrum yield and the total yield of IgG, protein and lactose were higher (p ≤ 0.05) in the supplemented group. The calving assistance rate was lower (p ≤ 0.012), and the neonatal vitality score was higher (p ≤ 0.001) in the MgB group. Improved parameters related to cow health and fertility were observed in the supplemented group. The MgB group also had higher milk yield during the first week of lactation (p ≤ 0.001), and a higher (p ≤ 0.05) body condition score from 3 to 9 weeks after calving. In conclusion, prepartum MgB supplementation provides a wide range of benefits for dairy cows, as well as their newborn calves.

Continuum interpretation of mechano-adaptation in micropatterned epithelia informed by in vitro experiments.

Epithelial tissues adapt their form and function following mechanical perturbations, or mechano-adapt, and these changes often result in reactive forces that oppose the direction of the applied change. Tissues subjected to ectopic tensions, for example, employ behaviors that lower tension, such as increasing proliferation or actomyosin turnover. This oppositional behavior suggests that the tissue has a mechanical homeostasis. Whether attributed to maintenance of cellular area, cell density, or cell and tissue tensions, epithelial mechanical homeostasis has been implicated in coordinating embryonic morphogenesis, wound healing, and maintenance of adult tissues. Despite advances toward understanding the feedback between mechanical state and tissue response in epithelia, more work remains to be done to examine how tissues regulate mechanical homeostasis using epithelial sheets with defined micropatterned shapes. Here, we used cellular microbiaxial stretching (CμBS) to investigate mechano-adaptation in micropatterned tissues of different shape consisting of Madin-Darby canine kidney cells. Using the CμBS platform, tissues were subjected to a 30% stretch that was held for 24h. We found that, following stretch, tissue stresses immediately increased then slowly evolved over time, approaching their pre-stretch values by 24h. Organization of the actin cytoskeletal was found to play a role in this process: anisotropic ally structured tissues exhibited anisotropic stress patterns, and the cytoskeletal became more aligned following stretch and reorganized over time. Interestingly, in unstretched tissues, stresses also decreased, which was found to be driven by proliferation-induced cellular confinement and change in tissue thickness. We modeled these behaviors with a continuum-based model of epithelial growth that accounted for stress-induced actin remodeling and proliferation, and found this model to strongly capture experimental behavior. Ultimately, this combined experimental-modeling approach suggests that epithelial mechano-adaptation depends on cellular architecture and proliferation, which can be modeled with a field-averaged approach applicable to more specific contexts in which change is driven by epithelial mechanical homeostasis. Insight box Epithelial tissues adapt their form and function following mechanical perturbation, and it is thought that this 'mechano-adaptation' plays an important role in driving processes like embryonic morphogenesis, wound healing, and adult tissue maintenance. Here, we use cellular microbiaxial stretching to probe this process in vitro in small epithelial tissues whose geometries were both controlled and varied. By using a highly precise stretching device and a continuum mechanics modeling framework, we revealed that tissue mechanical state changes following stretch and over time, and that this behavior can be explained by stress-dependent changes in actin fibers and proliferation. Integration of these approaches enabled a systematic approach to empirically and precisely measure these phenomena.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells

Ethnopharmacological relevanceLemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. Materials and methodsWe analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. ResultsLMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 μg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. ConclusionLMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

Effect of cannabis smoke condensate on human nasal epithelial cell adhesion, growth, and migration

ObjectiveWhen inhaled, cannabis smoke interacts with airway tissues, including the nasal mucosa, which may lead to nasal pathologies. We examined the effect of cannabis smoke condensate (CSC) on nasal epithelial cell and tissue behaviors. MethodsHuman nasal epithelial cells were exposed or not to CSC at different concentrations (1, 5, 10, and 20 %) and for different durations. Cell adhesion and viability were assessed, as well as post-wound cell migration and lactate dehydrogenase (LDH) release. ResultsThe nasal epithelial cells showed a larger cell size and a faint nucleus following exposure to CSC, compared to that observed in that control. This was supported by fewer adherent cells present after exposure for either 1 or 24 h to 5, 15, and 20 % CSC. CSC also had a significant toxic effect by reducing cell viability after both 1 and 24 h of exposure. This toxic effect was significant even at a low concentration (1 %) of CSC. The effects on nasal epithelial cell viability were confirmed by the decrease in cell migration. After the scratch and subsequent exposure to CSC for either 6 or 24 h, a complete inhibition of nasal epithelial cell migration was observed, compared to that found in the controls. CSC was toxic to the nasal epithelial cells, as the level of LDH significantly increased following cell exposure all CSC concentrations. ConclusionCannabis smoke condensate had a negative effect on several nasal epithelial cell behaviors. These findings indicate that cannabis smoke could be a threat to nasal tissues and ultimately lead to nasal and sinus disorders.

Early weaning causes small intestinal atrophy by inhibiting the activity of intestinal stem cells: involvement of Wnt/β-catenin signaling

BackgroundEarly weaning and shorter breastfeeding duration are applied by a proportion of young mothers, especially in the social spheres of poverty-stricken areas. Early childhood is a critical period for intestinal development, which is driven by intestinal stem cells (ISCs). However, how early weaning practice affects the function of ISCs to mediate intestinal development remains unclear.MethodsWe established an excellent early weaning mice model that has significant intestinal atrophy and growth arrest symptoms to explore the responses of ISCs to early weaning. The primary and passaged intestinal organoids from the suckling or early weaning mice were cultured to explore the underlying mechanism of early weaning affecting the ISCs.ResultsEarly weaning depressed the self-renewal of ISCs and attenuated the activity of ISCs-driven intestinal epithelial regeneration and crypt expansion in vivo and ex-vivo. Further results showed that early weaning retarded the differentiation of ISCs into transit-amplifying cells and Paneth cells, and accelerated the apoptosis of villous epithelial cells, jointly leading to intestinal epithelial atrophy. Mechanistically, early weaning inhibited Wnt signaling in ISCs, while an exogenous Wnt amplifier restored ISCs’ function in ex-vivo.ConclusionOur findings indicate that early weaning depresses the activity of ISCs via attenuating Wnt/β-catenin signaling and triggers the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-17 in jejunum, thereby impeding ISCs-driven epithelial regeneration and intestinal growth, which may provide a basal theory for the development of infant nutrients targeting stem cells to alleviate early weaning-induced intestinal problems.

Abstract 1705: SPOP: An essential gene for normal and prostate tumor cells

Abstract Background: Prostate cancer (PC) is the single most common and second-most lethal cancer in men, with over 268,000 estimated cases and over 34,500 estimated deaths in the US in 2022. The Speckle-Type POZ protein (SPOP) mutant subclass of PC accounts for 10% to 15% of all primary PC cases. SPOP is an adaptor for Cullin3/Ring (CUL3-RING)-type E3 ubiquitin ligase complexes and provides substrate specificity. The Cancer Genome Atlas (TCGA) studies show that SPOP is the most frequently mutated gene in primary prostate cancer (PC). Interestingly, PC-associated SPOP mutations are always missense and occur in a heterozygous fashion. The current gap in knowledge is the lack of understanding of the role of wildtype SPOP in PC. Methods: By utilizing prostate specific SPOP knockout (KO) mice, we recently reported increased levels of AR and MYC protein and increased cellular turnover (both proliferation and apoptosis) in the prostate luminal epithelium compared to wildtype prostates. We now characterized these mice for the expression of Cre protein and SPOP mRNA at different age using immunohistochemistry and RNA in situ hybridization. Furthermore, we performed RNA-sequencing analysis in the SPOP knockout mice and matched control littermates. Moreover, we performed RNA-seq in LNCaP, LNCaP-Abl, and RWPE1 cells following SPOP inhibition via siRNA targeting SPOP. Finally, we compared our SPOP inhibition signature from in vitro cell lines and prostate specific SPOP knockout murine model to gain insights about the role of wildtype SPOP protein in the prostate epithelium. Result: Using our Spopfl/fl;PBCre+ model, we observed SPOP floxed cells are rapidly lost and the murine prostate epithelium was repopulated with SPOP wildtype carrying cells. Similarly, knockdown (KD) of SPOP through siRNA treatment in a panel of PC cell lines resulted in a significant reduction in cell viability. These observations suggest that SPOP is important for the normal prostate cell viability. Further transcriptomic profiling of SPOP KO (from transgenic murine model) as well as siSPOP treated in vitro prostate cell lines revealed a significant reduction in the transcriptional activity of the AR. Conclusion: Our data illustrate for the first time a critical role for SPOP in the growth and survival of the prostate epithelium and prostate cancer cell. Our findings further validates SPOP as a important therapeutic target for the treatment of prostate cancer. Citation Format: Kinza Rizwan, Darlene Skapura, Cammy Mason, Cristian Coarfa, Nicholas Mitsiades, Damian Young, Salma Kaochar. SPOP: An essential gene for normal and prostate tumor cells [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 1705.

Abstract 6776: Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC

Abstract Background: While cancer therapies are becoming increasingly more targeted, our understanding of the genomic changes occurring during these treatments is lacking. Moreover, the tumoral heterogeneity and its spatial distribution remain important obstacles in predicting tumor evolution and treatment responses. In recent years, genomic spatial profiling techniques have shed light on this complexity by detecting gene expression changes in heterogenous tumor regions and their microenvironment over the course of treatment. Methods: Tepotinib is a highly selective and potent MET class Ib inhibitor, approved for the treatment of patients with NSCLC with MET exon 14 skipping alterations. To detect genomic changes during neoadjuvant treatment with tepotinib, a GeoMx® digital spatial profiler (DSP; Nanostring Technologies) analysis was conducted in paired baseline and on-treatment patient samples. GeoMx® DSP was performed by selecting regions of interest with nuclear, TTF-1, CD3 and CD33 morphology markers followed by next-generation sequencing (NGS) using the Cancer Transcriptome Atlas RNA panel (>1,800 cancer-related genes). Results: Following a poor response to chemotherapy, neoadjuvant tepotinib treatment was initiated in a 54-year-old female, never-smoker with NSCLC harboring a MET exon 14 alteration. Owing to a good response to tepotinib, surgical resection of the tumor was decided by the tumor board given the downgrading (from IIIB to IA1) and resulting in a major pathological response. This resection allowed spatial transcriptomics to be compared to a baseline biopsy from the same patient. MET signaling inhibition by tepotinib treatment increased immune cell infiltration in the tumor microenvironment compared to the baseline sample. This enhanced tumoral immune cell density was shown by the fluorescent morphology markers and confirmed through spatial deconvolution of the NGS results. It was further characterized by the significantly altered gene expression levels between the two samples. Gene ontology (GO) assessment of the differentially expressed genes revealed GO terms associated with tumorigenesis in the basal sample, such as cell mobility and motility, vascular epithelial growth factor production and cell communication. In turn, the tepotinib-treated tissue mainly exhibited tumor-clearing characteristics, including antigen processing and presentation through major histocompatibility complex classes I & II, T cell-mediated toxicity, and granulocyte and leukocyte activation. Conclusion: This is the first spatial genomic characterization of the effect of tepotinib on the tumor and its microenvironment in a patient with NSCLC over time. Results from this case report highlight the impact of MET inhibition by tepotinib on the tumor immune microenvironment, likely enabling the recruitment of antigen-presenting cells for tumor clearance, while reducing the expression of tumor-promoting genes. Citation Format: Manon A. Simard, Felix Geist, Carlos Cabrera, Santiago Viteri, Joachim Albers, Michael Zuehlsdorf, Niki Karachaliou. Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC. [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 6776.

Defining morphologic features of invasion in pulmonary non-mucinous adenocarcinoma with lepidic growth - A proposal by the IASLC Pathology Committee.

Since the 8th edition of UICC/AJCC TNM classification system the primary tumor pT stage is determined based on presence and size of the invasive components. The aim of this study was to identify histological features in tumors with lepidic growth pattern that may be used to establish criteria for distinguishing invasive from non-invasive areas. A Delphi approach was used with two rounds of blinded anonymized analysis of resected non-mucinous lung adenocarcinoma cases with presumed invasive and non-invasive components, followed by one round of reviewer de-anonymized and unblinded review of cases with known outcomes. A digital pathology platform was used for measuring total tumor size and invasive tumor size. The mean coefficient of variation for measuring total tumor size and tumor invasive size was 6.9% (range 1.7-22.3%) and 54% (range 14.7-155%), respectively, with substantial variations in interpretation of the size and location of invasion among pathologists. Following the presentation of the results and further discussion among members at large of the IASLC Pathology Committee, extensive epithelial proliferation (EEP) in areas of collapsed lepidic growth pattern is recognized as a feature likely to be associated with invasive growth. EEP is characterized by multilayered luminal epithelial cell growth, usually with high grade cytological features in several alveolar spaces. Collapsed alveoli and transition zones with EEP were identified by the Delphi process as morphologic features that were a source of interobserver variability. Definition criteria for collapse and EEP are proposed to improve reproducibility of invasion measurement.

Multi-omics fusion analysis models with machine learning predict survival of HER2-negative metastatic breast cancer: a multicenter prospective observational study.

Multi-omics fusion analysis models with machine learning predict survival of HER2-negative metastatic breast cancer: a multicenter prospective observational study.

Coordination of tissue homeostasis and growth by the Scribble-α-Catenin-Septate junction complex

Maintaining apicobasal polarity (ABP) is crucial for epithelial integrity and homeostasis during tissue development. Although intracellular mechanisms underlying ABP establishment have been well studied, it remains to be addressed how theABP coordinates tissue growth and homeostasis. By studying Scribble, a key ABP determinant, we address molecular mechanisms underlying ABP-mediated growth control in the Drosophila wing imaginal disc. Our data reveal that genetic and physical interactions between Scribble, Septate junction complex and α-Catenin appear to be key for sustaining ABP-mediated growth control. Cells with conditional scribble knockdown instigate the loss of α-Catenin, ultimately leading to the formation of neoplasia accompanying with activation of Yorkie. In contrast, cells expressing wild type scribble progressively restore ABP in scribble hypomorphic mutant cells in a non-autonomous manner. Our findings provide unique insights into cellular communication among optimal and sub-optimal cells to regulate epithelial homeostasis and growth.

Influence of Surface Characteristics of TiO2 Coatings on the Response of Gingival Cells: A Systematic Review of In Vitro Studies.

The soft tissue-implant interface requires the formation of epithelium and connective tissue seal to hinder microbial infiltration and prevent epithelial down growth. Nanoporous titanium dioxide (TiO2) surface coatings have shown good potential for promoting soft tissue attachment to implant surfaces. However, the impact of their surface properties on the biological response of gingival cells needs further investigation. This systematic review aimed to investigate the cellular behavior of gingival cells on TiO2-implant abutment coatings based on in vitro studies. The review was performed to answer the question: "How does the surface characteristic of TiO2 coatings influence the gingival cell response in in vitro studies?". A search in MEDLINE/PubMed and the web of science databases from 1990 to 2022 was performed using keywords. A quality assessment of the studies selected was performed using the SciRAP method. A total of 11 publications were selected from the 289 studies that fulfilled the inclusion criteria. The mean reporting and methodologic quality SciRAP scores were 82.7 ± 6.4/100 and 87 ± 4.2/100, respectively. Within the limitations of this in vitro systematic review, it can be concluded that the TiO2 coatings with smooth nano-structured surface topography and good wettability improve gingival cell response compared to non-coated surfaces.

Live Cells versus Fixated Cells: Kinetic Measurements of Biomolecular Interactions with the LigandTracer Method and Surface Plasmon Resonance Microscopy.

Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.

Frequency of Her2-low in colorectal cancer and its relations with the tumor microenvironment

BackgroundTo date, little is known regarding human epithelial growth factor receptor (HER2) low-expressing colorectal cancer (CRC). Due to promising rising therapies with HER2-antibody-drug conjugates we aimed to analyze the frequency of HER2-low in patients with CRC. Additionally we characterized the clinicopathologic background of this group and its potential relationship with the tumor microenvironment represented by budding and tumor infiltrating lymphocytes (TILs). Methods319 patients with CRC, stages I–IV, were enrolled. HER2-immunohistochemistry (IHC) as well as fluorescence in situ hybridization (FISH) were performed on tissue microarrays. IHC was evaluated semiquantitatively and software-assisted using the HERACLES Diagnostic Criteria for CRC. HER2-low was defined as IHC 1 + or 2 +/FISH negative. HER2-IHC results were compared with budding, TILs and their combinations. ResultsThe HER2 low-expressing subset represented almost one half of all CRC (47.1 %). Assessment was highly reproducible with different methods. HER2-low cases were significantly more often lower T-, N-, and tumor stage and had less L1 compared with HER2-0. Additionally, they showed more often TILs > 5 % (p = 0.001). The difference between HER2-0 and HER2-low was highly significant between the four budding/TILs-groups (p < 0.001). Cases with low budding/high TILs were more often HER2-low. The highest difference was seen between the low budding/high TILs-group and the low budding/low TILs-group (p < 0.001). ConclusionsHER2-low expression in CRC is frequent and involves nearly one half of all patients. We could show a relationsship between HER2-low expression and the tumor microenvironment. Special attention should be paid to the low budding/high TILs group in future research.

Purriato is a conserved small open reading frame gene that interacts with the CASA pathway to regulate muscle homeostasis and epithelial tissue growth in Drosophila.

Recent advances in proteogenomic techniques and bioinformatic pipelines have permitted the detection of thousands of translated small Open Reading Frames (smORFs), which contain less than 100 codons, in eukaryotic genomes. Hundreds of these actively translated smORFs display conserved sequence, structure and evolutionary signatures indicating that the translated peptides could fulfil important biological roles. Despite their abundance, only tens of smORF genes have been fully characterised; these act mainly as regulators of canonical proteins involved in essential cellular processes. Importantly, some of these smORFs display conserved functions with their mutations being associated with pathogenesis. Thus, investigating smORF roles in Drosophila will not only expand our understanding of their functions but it may have an impact in human health. Here we describe the function of a novel and essential Drosophila smORF gene named purriato (prto). prto belongs to an ancient gene family whose members have expanded throughout the Protostomia clade. prto encodes a transmembrane peptide which is localized in endo-lysosomes and perinuclear and plasma membranes. prto is dynamically expressed in mesodermal tissues and imaginal discs. Targeted prto knockdown (KD) in these organs results in changes in nuclear morphology and endo-lysosomal distributions correlating with the loss of sarcomeric homeostasis in muscles and reduction of mitosis in wing discs. Consequently, prto KD mutants display severe reduction of motility, and shorter wings. Finally, our genetic interaction experiments show that prto function is closely associated to the CASA pathway, a conserved mechanism involved in turnover of mis-folded proteins and linked to muscle dystrophies and neurodegenerative diseases. Thus, this study shows the relevance of smORFs in regulating important cellular functions and supports the systematic characterisation of this class of genes to understand their functions and evolution.

ZnO nanoparticles-modified polycaprolactone-gelatin membranes for guided/bone tissue regeneration, antibacterial and osteogenic differentiation properties

Periodontitis is a highly prevalent infectious disease that causes the progressive destruction of the periodontal supporting tissues. If left untreated, it can lead to tooth loss impairing oral function, aesthetics, and the patient’s overall quality of life. Guided and Bone Tissue Regeneration (GTR/BTR) are surgical therapies based on the placement of a membrane that prevents epithelial growth into the defect, allowing the periodontal/bone cells (including stem cells) to regenerate or restore the affected tissues. The success of these therapies is commonly affected by the local bacterial colonization of the membrane area and its fast biodegradation, causing postoperative infections and a premature rupture of the membrane limiting the regeneration process. This study presents the antibacterial and osteogenic differentiation properties of polycaprolactone-gelatin (PCL-G) electrospun membranes modified with ZnO nanoparticles (ZnO-NPs). The membranes´ chemical composition, surface roughness, biodegradation, water wettability, and mechanical properties under simulated physiological conditions, were analyzed by the close relationship with their biological properties. The PCL-G membranes modified with 1, 3, and 6% w/w of ZnO-NPs showed a significant reduction in the planktonic and biofilm formation of four clinically relevant bacteria; A. actinomycetemcomitans serotype b, P. gingivalis, E. coli, and S. epidermidis. Additionally, the membranes presented appropriate mechanical properties and biodegradation rates to be potentially used in clinical treatments. Notably, the membranes modified with the lowest concentration of ZnO-NPs (1% w/w) stimulated the production of osteoblast markers and calcium deposits in human bone marrow-derived mesenchymal stem cells (BM-MSC) and were biocompatible to human osteoblasts cells (hFOB). These results suggest that the PCL-G membranes with 1% w/w of ZnO-NPs are high-potential candidates for GTR/BTR treatments, as they were the most effective in terms of better antibacterial effectiveness at a lower NPs-concentration while creating a favorable cellular microenvironment for bone growth.