The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

Revitalizing the skin: Exploring the role of barrier repair moisturizers.

BACKGROUND: Moisturizers are designed to maintain skin health and treat dermatological conditions associated with impaired skin barrier function. However, differences in their composition account for the differences in their effect. AIMS: This narrative review aims to discuss the role of barrier repair moisturizers, highlight the role of different components in a moisturizer and their role in impaired skin conditions (e.g., dry, itchy, inflamed, sensitive skin, atopic eczema), and thereby empower dermatologists and pediatricians in selecting the right moisturizer. METHODS: PubMed, Embase, and Scopus electronic databases were searched from January 2000 to June 2023 for publications on skin barrier repair and use of barrier repair moisturizers for the treatment of dry, itchy, inflamed, sensitive skin, or atopic eczema. Studies conducted in humans, published in English for which full texts were freely available were included. RESULTS: The structure and composition of lipid lamellae within the stratum corneum play an important role in maintaining an effective skin barrier and protecting the body from various external assaults. Endocannabinoid mediators play an active role in maintaining skin barrier function. Moisturizers containing physiological lipids and functional ingredients (e.g., endocannabinoids such as palmitoylethanolamide [PEA]) and based on the principles of biomimic technology are demonstrated to be beneficial for the management of conditions associated with a disrupted skin barrier. CONCLUSIONS: Moisturizer based on the innovative biomimic formulation has good cosmetic efficacy and is generally well tolerated, and the addition of PEA might represent a new generation of compounds that may be beneficial for long-term management of impaired skin conditions.

IGF-1R targeting in cancer - does sub-cellular localization matter?

The insulin-like growth factor receptor (IGF-1R) was among the most intensively pursued kinase targets in oncology. However, even after a slew of small-molecule and antibody therapeutics reached clinical trials for a range of solid tumors, the initial promise remains unfulfilled. Mechanisms of resistance to, and toxicities resulting from, IGF-1R-targeted drugs are well-catalogued, and there is general appreciation of the fact that a lack of biomarker-based patient stratification was a limitation of previous clinical trials. But no next-generation therapeutic strategies have yet successfully exploited this understanding in the clinic.Currently there is emerging interest in re-visiting IGF-1R targeted therapeutics in combination-treatment protocols with predictive biomarker-driven patient-stratification. One such biomarker that emerged from early clinical trials is the sub-cellular localization of IGF-1R. After providing some background on IGF-1R, its drugging history, and the trials that led to the termination of drug development for this target, we look more deeply into the correlation between sub-cellular localization of IGF-1R and susceptibility to various classes of IGF-1R - targeted agents.

Molecularly Defined Subsets of Ewing Sarcoma Tumors Differ in Their Responses to IGF1R and WEE1 Inhibition.

PURPOSE: Targeted cancer therapeutics have not significantly benefited patients with Ewing sarcoma with metastatic or relapsed disease. Understanding the molecular underpinnings of drug resistance can lead to biomarker-driven treatment selection. EXPERIMENTAL DESIGN: Receptor tyrosine kinase (RTK) pathway activation was analyzed in tumor cells derived from a panel of Ewing sarcoma tumors, including primary and metastatic tumors from the same patient. Phospho-RTK arrays, Western blots, and IHC were used. Protein localization and the levels of key markers were determined using immunofluorescence. DNA damage tolerance was measured through PCNA ubiquitination levels and the DNA fiber assay. Effects of pharmacologic inhibition were assessed in vitro and key results validated in vivo using patient-derived xenografts. RESULTS: Ewing sarcoma tumors fell into two groups. In one, IGF1R was predominantly nuclear (nIGF1R), DNA damage tolerance pathway was upregulated, and cells had low replication stress and RRM2B levels and high levels of WEE1 and RAD21. These tumors were relatively insensitive to IGF1R inhibition. The second group had high replication stress and RRM2B, low levels of WEE1 and RAD21, membrane-associated IGF1R (mIGF1R) signaling, and sensitivity to IGF1R or WEE1-targeted inhibitors. Moreover, the matched primary and metastatic tumors differed in IGF1R localization, levels of replication stress, and inhibitor sensitivity. In all instances, combined IGF1R and WEE1 inhibition led to tumor regression. CONCLUSIONS: IGF1R signaling mechanisms and replication stress levels can vary among Ewing sarcoma tumors (including in the same patient), influencing the effects of IGF1R and WEE1 treatment. These findings make the case for using biopsy-derived predictive biomarkers at multiple stages of Ewing sarcoma disease management.

Safety and clinical outcomes of remdesivir in hospitalised COVID-19 patients: a retrospective analysis of active surveillance database.

BACKGROUND: Real-world data on safety and clinical outcomes of remdesivir in COVID-19 management is scant. We present findings of data analysis conducted for assessing the safety and clinical outcomes of remdesivir treatment for COVID-19 in India. METHODS: This retrospective analysis used data from an active surveillance programme database of hospitalised patients with COVID-19 who were receiving remdesivir. RESULTS: Of the 2329 patients included, 67.40% were men. Diabetes (29.69%) and hypertension (20.33%) were the most common comorbidities. At remdesivir initiation, 2272 (97.55%) patients were receiving oxygen therapy. Remdesivir was administered for 5 days in 65.38% of patients. Antibiotics (64.90%) and steroids (47.90%) were the most common concomitant medications. Remdesivir was overall well tolerated, and total 119 adverse events were reported; most common were nausea and vomiting in 45.40% and increased liver enzymes in 14.28% patients. 84% of patients were cured/improved, 6.77% died and 9.16% showed no improvement in their clinical status at data collection. Subgroup analyses showed that the mortality rate was significantly lower in patients < 60 years old than in those > 60 years old. Amongst patients on oxygen therapy, the cure/improvement rate was significantly higher in those receiving standard low-flow oxygen than in those receiving mechanical ventilation, non-invasive ventilation, or high-flow oxygen. Factors that were associated with higher mortality were age > 60 years, cardiac disease, diabetes high flow oxygen, non-invasive ventilation and mechanical ventilation. CONCLUSION: Our analysis showed that remdesivir is well tolerated and has an acceptable safety profile. The clinical outcome of cure/improvement was 84%, with a higher improvement in patients < 60 years old and on standard low-flow oxygen.

The emerging role of mycophenolate mofetil in interstitial lung diseases.

INTRODUCTION: Mycophenolate mofetil (MMF), initially approved to prevent rejection in solid organ allograft, is now being increasingly used for other conditions. Over the last decade, MMF has emerged as a useful therapy for a variety of immune-mediated diseases. AREAS COVERED: There has been a growing interest in the clinical use of MMF in the treatment of ILDs due to its versatile anti-inflammatory, immunomodulatory, anti-fibrotic and anti-proliferative properties. In this focussed review, we summarize the available literature using the Pubmed, Science Direct and EMBASE databases published until June 2021 on the efficacy and tolerability of MMF in various ILDs. EXPERT OPINION: Other than idiopathic pulmonary fibrosis (IPF) and its broader category of progressive fibrosing ILD, there have been no drugs approved by relevant regulatory agencies for the treatment of the multiple other forms of ILD. Though results are limited, immunosuppressants such as MMF have shown promise as an effective and well-tolerated steroid-sparing agent, providing hope that the limited treatment armamentarium for ILDs can be expanded.

The Eyes Absent proteins in development and in developmental disorders.

The Eyes Absent (EYA) transactivator-phosphatase proteins are important contributors to cell-fate determination processes and to the development of multiple organs. The transcriptional regulatory activity as well as the protein tyrosine phosphatase activities of the EYA proteins can independently contribute to proliferation, differentiation, morphogenesis and tissue homeostasis in different contexts. Aberrant EYA levels or activity are associated with numerous syndromic and non-syndromic developmental disorders, as well as cancers. Commensurate with the multiplicity of biochemical activities carried out by the EYA proteins, they impact upon a range of cellular signaling pathways. Here, we provide a broad overview of the roles played by EYA proteins in development, and highlight the molecular signaling pathways known to be linked with EYA-associated organ development and developmental disorders.

Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia.

BACKGROUND: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. METHODS: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy. RESULTS: Foxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. CONCLUSIONS: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

The Eyes Absent Proteins: Unusual HAD Family Tyrosine Phosphatases.

Here, we review the haloacid dehalogenase (HAD) class of protein phosphatases, with a particular emphasis on an unusual group of enzymes, the eyes absent (EYA) family. EYA proteins have the unique distinction of being structurally and mechanistically classified as HAD enzymes, yet, unlike other HAD phosphatases, they are protein tyrosine phosphatases (PTPs). Further, the EYA proteins are unique among the 107 classical PTPs in the human genome because they do not use a Cysteine residue as a nucleophile in the dephosphorylation reaction. We will provide an overview of HAD phosphatase structure-function, describe unique features of the EYA family and their tyrosine phosphatase activity, provide a brief summary of the known substrates and cellular functions of the EYA proteins, and speculate about the evolutionary origins of the EYA family of proteins.

The emerging role of nebulization for maintenance treatment of chronic obstructive pulmonary disease at home.

Inhalation therapy is the cornerstone of chronic obstructive pulmonary disease (COPD) management. However, for many COPD patients who are managed at home, nebulization therapy offers an effective alternative treatment and fulfills the gap of catering to the specific population of patients who are unable to use handheld inhaler devices appropriately. The present review highlights key aspects, namely selection of the right beneficiaries for home nebulization, available drugs in nebulized formulations for the treatment of COPD, and the importance of care, cleaning, and maintenance, which are prerequisites for ensuring successful nebulization therapy.

Targeting EYA3 in Ewing Sarcoma Retards Tumor Growth and Angiogenesis.

EWSR1/FLI1, the most common fusion gene in Ewing sarcoma, upregulates expression of the Eyes Absent 3 (EYA3) transactivator-phosphatase protein. The purpose of this study was to investigate molecular and cellular mechanisms through which EYA3 might promote Ewing sarcoma tumor growth and to determine whether the EYA3 tyrosine phosphatase activity represents a viable therapeutic target. We used genetic and pharmacologic modulation of EYA3 in cell line-based xenografts to examine how loss of EYA3 tyrosine phosphatase activity affects tumor growth and angiogenesis. Molecular mechanisms were evaluated in vivo and in vitro through analyses of tumor tissue and multicellular tumor spheroids. Our results show that both loss of EYA3 in Ewing sarcoma cells and pharmacologic inhibition of the EYA3 tyrosine phosphatase activity inhibit tumor growth and tumor angiogenesis. EYA3 regulates levels of VEGFA in Ewing tumors, as well as promoting DNA damage repair and survival of Ewing sarcoma tumor cells. Target engagement is demonstrated in tumor tissue through elevated levels of the EYA3 substrate H2AX-pY142 upon loss of EYA3 or with Benzarone treatment. The efficacy of EYA3 tyrosine phosphatase inhibition in attenuating tumor growth and angiogenesis is corroborated in an Ewing sarcoma patient-derived tumor xenograft. Together, the results presented here validate EYA3 as a target for the development of novel Ewing sarcoma therapeutic strategies, and set the stage for evaluating the efficacy of combining the antiangiogenic and anti-cell survival effects of EYA3 inhibition with cytotoxic chemotherapy.

The multi-functional eyes absent proteins.

The Eyes Absent (EYA) proteins are the only known instance of a single polypeptide housing the following three separable biochemical activities: tyrosine phosphatase, threonine phosphatase, and transactivation. This uniquely positions the EYAs to participate in both transcriptional regulation and signal transduction pathways. But it also complicates the assignment of biological roles to individual biochemical activities through standard loss-of-function experiments. Nevertheless, there is an emerging literature linking developmental and pathological functions with the various EYA activities, and a growing list of disease states that might benefit from EYA-targeted therapeutics. There also remain multiple unresolved issues with significant implications for our understanding of how the EYAs might impact such ubiquitous signaling cascades as the MYC and Notch pathways. This review will describe the unique juxtaposition of biochemical activities in the EYAs, their interaction with signaling pathways and cellular processes, emerging evidence of roles in disease states, and the feasibility of therapeutic targeting of individual EYA activities. We will focus on the phosphatase activities of the vertebrate EYA proteins and will examine the current state of knowledge regarding: • substrates and signaling pathways affected by the EYA tyrosine phosphatase activity; • modes of regulation of the EYA tyrosine phosphatase activity; • signaling pathways that implicate the threonine phosphatase activity of the EYAs including a potential interaction with PP2A-B55α; • the interplay between the two phosphatase activities and the transactivation function of the EYAs; • disease states associated with the EYAs and the current state of development of EYA-targeted therapeutics.

A Study to Evaluate the Efficacy of an 810-nm Diode Laser in the Maintenance of Dental Implants: A Peri-Implant Sulcular Fluid Analysis.

Biological implant failures are primarily related to biofilm, which can lead to peri-mucositis and, further on, peri-implantitis. The 810-nm diode laser has an affinity for pigmented chromophores, so its use in the peri-implant sulcus has a significant bactericidal effect on the black-pigmented anaerobes such as Porphyromonas gingivalis. Therefore, it can be used to eliminate or reduce the bacterial count in the peri-implant sulcular fluid (PISF), thus increasing the life of the implants and reducing the chances of failure. The purpose of this study was to evaluate the efficacy of the 810-nm diode laser for the maintenance of dental implants and its use as a regular in-office tool for limiting the microbiological count in the PISF. Twenty patients undergoing implant treatment at the Department of Periodontology and Oral Implantology were randomly selected for the study. PISF samples were collected before and after the sulcus was lased with an 810-nm diode laser and sent for quantitative microbiological analysis using universal bacterial count, and the quantity of P gingivalis was evaluated using real-time polymerase chain reaction (PCR). The analysis revealed that after diode application, the median percentage drop in the microbial count was 76.67% and the median percentage drop in P gingivalis count was 99.28%. The use of an 810-nm diode laser resulted in the following outcomes: (1) drastic reduction in the total bacterial count around the implant and (2) significant reduction in the P gingivalis count, as evaluated by real-time PCR.

The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis.

Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrest and elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.

Real-world evaluation of the clinical safety and efficacy of fluticasone/formoterol FDC via the Revolizer® in patients with persistent asthma in India.

The combination of inhaled corticosteroids (ICS) and long-acting beta2-agonists (LABAs) is widely used for the management of asthma. This prospective, open-label, non-comparative, observational, 24-week multicentre study is the first real-world study from India to compare the efficacy and safety of fixed-dose combination of fluticasone/formoterol (Maxiflo® 100/6 mcg or 250/6 mcg) capsules via the Revolizer® device in patients with persistent asthma. The primary efficacy analyses included mean change in Asthma Control Test (ACT™) at 4, 8, 16 and 24 weeks. Secondary efficacy analyses included mean change in morning and evening peak expiratory flow rate (PEFR) at the end of 4, 8, 16 and 24 weeks, number of patients having symptom-free days and nights at the end of 4, 8, 16 and 24 weeks, the number and severity of exacerbations over 24 weeks and response to the Usability Preference Satisfaction Confidence questionnaire after 1 week. Overall, 385 (of 401; 96.01%) enrolled patients completed the study. The mean change in ACT™ score was 6.7 ± 3.71 (95% CI: 6.32, 7.06; p < 0.0001) at week 24. The ACT™ score at weeks 4, 8 and 16 showed progressive and statistically significant increase from baseline. A statistically significant improvement in morning and evening PEFR at weeks 4, 8, 16 and 24 was reported. The proportion of patients experiencing symptom-free days and nights continuously increased from baseline to week 24. A good safety profile over the 24-week period was observed. The Revolizer® device was preferred by 94.26% patients over their current device. Fluticasone propionate/formoterol fumarate FDC capsules administered via a single-dose dry powder inhaler ([DPI], (Revolizer®) offers a novel, well-tolerated and effective treatment option for the long-term management of asthma.

The EYA3 tyrosine phosphatase activity promotes pulmonary vascular remodeling in pulmonary arterial hypertension.

In pulmonary hypertension vascular remodeling leads to narrowing of distal pulmonary arterioles and increased pulmonary vascular resistance. Vascular remodeling is promoted by the survival and proliferation of pulmonary arterial vascular cells in a DNA-damaging, hostile microenvironment. Here we report that levels of Eyes Absent 3 (EYA3) are elevated in pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension and that EYA3 tyrosine phosphatase activity promotes the survival of these cells under DNA-damaging conditions. Transgenic mice harboring an inactivating mutation in the EYA3 tyrosine phosphatase domain are significantly protected from vascular remodeling. Pharmacological inhibition of the EYA3 tyrosine phosphatase activity substantially reverses vascular remodeling in a rat model of angio-obliterative pulmonary hypertension. Together these observations establish EYA3 as a disease-modifying target whose function in the pathophysiology of pulmonary arterial hypertension can be targeted by available inhibitors.

An opsin 5-dopamine pathway mediates light-dependent vascular development in the eye.

During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.

The Protein Tyrosine Phosphatase Activity of Eyes Absent Contributes to Tumor Angiogenesis and Tumor Growth.

DNA damage repair capacity is required for cells to survive catastrophic DNA damage and proliferate under conditions of intratumoral stress. The ability of the minor histone protein H2AX to serve as a hub for the assembly of a productive DNA damage repair complex is a necessary step in preventing DNA damage-induced cell death. The Eyes Absent (EYA) proteins dephosphorylate the terminal tyrosine residue of H2AX, thus permitting assembly of a productive DNA repair complex. Here, we use genetic and chemical biology approaches to separately query the roles of host vascular endothelial cell and tumor cell EYA in tumor growth. Deletion of Eya3 in host endothelial cells significantly reduced tumor angiogenesis and limited tumor growth in xenografts. Deletion of Eya3 in tumor cells reduced tumor cell proliferation and tumor growth without affecting tumor angiogenesis. A chemical inhibitor of the EYA tyrosine phosphatase activity inhibited both tumor angiogenesis and tumor growth. Simultaneously targeting the tumor vasculature and tumor cells is an attractive therapeutic strategy because it could counter the development of the more aggressive phenotype known to emerge from conventional antiangiogenic agents. Mol Cancer Ther; 17(8); 1659-69. ©2018 AACR.

Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids.

Under hypoxic conditions, tumor cells undergo a series of adaptations that promote evolution of a more aggressive tumor phenotype including the activation of DNA damage repair proteins, altered metabolism, and decreased proliferation. Together these changes mitigate the negative impact of oxygen deprivation and allow preservation of genomic integrity and proliferative capacity, thus contributing to tumor growth and metastasis. As a result the presence of a hypoxic microenvironment is considered a negative clinical feature of many solid tumors. Hypoxic niches in tumors also represent a therapeutically privileged environment in which chemo- and radiation therapy is less effective. Although the negative impact of tumor hypoxia has been well established, the precise effect of oxygen deprivation on tumor cell behavior, and the molecular signals that allow a tumor cell to survive in vivo are poorly understood. Multicellular tumor spheroids (MCTS) have been used as an in vitro model for the avascular tumor niche, capable of more accurately recreating tumor genomic profiles and predicting therapeutic response. However, relatively few studies have used MCTS to study the molecular mechanisms driving tumor cell adaptations within the hypoxic tumor environment. Here we will review what is known about cell proliferation, DNA damage repair, and metabolic pathways as modeled in MCTS in comparison to observations made in solid tumors. A more precise definition of the cell populations present within 3D tumor models in vitro could better inform our understanding of the heterogeneity within tumors as well as provide a more representative platform for the testing of therapeutic strategies.