Additional Layer Research Articles

Contrasting new and available reference genomes to highlight uncertainties in assemblies and areas for future improvement: an example with monodontid species

BackgroundReference genomes provide a foundational framework for evolutionary investigations, ecological analysis, and conservation science, yet uncertainties in the assembly of reference genomes are difficult to assess, and by extension rarely quantified. Reference genomes for monodontid cetaceans span a wide spectrum of data types and analytical approaches, providing the context to derive broader insights related to discrepancies and regions of uncertainty in reference genome assembly. We generated three beluga (Delphinapterus leucas) and one narwhal (Monodon monoceros) reference genomes and contrasted these with published chromosomal scale assemblies for each species to quantify discrepancies associated with genome assemblies.ResultsThe new reference genomes achieved chromosomal scale assembly using a combination of PacBio long reads, Illumina short reads, and Hi-C scaffolding data. For beluga, we identified discrepancies in the order and orientation of contigs in 2.2–3.7% of the total genome depending on the pairwise comparison of references. In addition, unsupported higher order scaffolding was identified in published reference genomes. In contrast, we estimated 8.2% of the compared narwhal genomes featured discrepancies, with inversions being notably abundant (5.3%). Discrepancies were linked to repetitive elements in both species.ConclusionsWe provide several new reference genomes for beluga (Delphinapterus leucas), while highlighting potential avenues for improvements. In particular, additional layers of data providing information on ultra-long genomic distances are needed to resolve persistent errors in reference genome construction. The comparative analyses of monodontid reference genomes suggested that the three new reference genomes for beluga are more accurate compared to the currently published reference genome, but that the new narwhal genome is less accurate than one published. We also present a conceptual summary for improving the accuracy of reference genomes with relevance to end-user needs and how they relate to levels of assembly quality and uncertainty.

Uniform Li-metal growth on renewable lignin with lithiophilic functional groups derived from wood for high-performance Li-metal batteries

Lithium metal batteries (LMBs) are promising energy storage devices with high energy density owing to their high theoretical capacity (3860 mAh g−1) and low reduction potential of lithium (Li)-metal anodes. However, their practical application is limited by poor Coulombic efficiency (CE) and safety issues due to Li dendrite growth during Li plating/stripping. Herein, a three-dimensional (3D) structure of lignin with a lithiophilic functional group derived from waste wood is suggested as an additional functional layer of LMBs, i.e., a lignosulfonic acid sodium salt (LASS) with a negatively charged sulfonate group (SO3−) in its chemical structure. The SO3− group enhances the Li-ion (Li+) affinity and enables Li+-flux control, supplying homogeneous Li growth sites. It induces stable electrochemical dendrite-free Li growth. Consequently, in the LASS-Cu||Li half-cell, a high CE of 96.7 % at 210 cycles and stable operation is achieved without a short circuit and, symmetric-cell with LASS layers shows stable cycling over 2000 h. Moreover, full-cells with LiFePO4 (LFP) and LiNi0.8Co0.1Mn0.1O2 (NCM 811) cathodes exhibit enhanced electrochemical performance. Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) reveal that the LASS layer suppressed dendritic Li growth and stimulated densely packed Li growth. This work suggests an effective strategy for improving the performance of LMBs using renewable lignin materials.

Effect of process parameters on mechanical properties and wettability of polylactic acid by fused filament fabrication process

Abstract Fiber-reinforced composites have changed additive manufacturing into a sustainable manufacturing paradigm with the capacity to produce items with outstanding mechanical performance. The fused filament fabrication (FFF) process is the latest in advanced digital fabrication techniques used for fabricating polymer material using additive layer deposition. In this study, the results of mechanical and vibration tests were used to examine the impact of process variables such as layer thickness (0.08 mm, 0.25 mm, and 0.64 mm), infill density (20 %, 40 %, 60 %, and 80 %), and infill pattern (rectilinear, triangular, and hexagonal). The novelty of this work is to correlate the contact angle measurement with the mechanical properties of the 3D-printed specimens. The adhesion behaviour of the 3D printed parts is examined by measuring the contact angle from the wettability test. From the findings, it was observed that the infill density and layer pattern play a significant role in the interlayer adhesion, as evident from the contact angle measurement.

Key factors in the decision-making process for mastectomy alone or breast reconstruction: A qualitative analysis

Breast cancer is the most common cancer to affect New Zealand women. Women diagnosed face several decisions regarding surgical treatment, including whether to undergo lumpectomy, mastectomy, or breast reconstruction. Reconstructive surgery adds an additional layer of complexity, with several reconstructive options, each associated with differing surgical and recovery times. Furthermore, surgical decisions are often made under time-pressure and significant diagnostic distress, therefore provision of good information to support decision-making is crucial to adequately inform women of their options. We interviewed 24 women who had undergone breast surgery within the preceding 12 months to assess the key factors leading to their decision to opt for their chosen surgical procedure. Interviews revealed that decision-making was complex and involved multiple factors. Women were ultimately confronted with assessing feminine identity versus survival. Whether opting for breast reconstruction or not, women were fearful of what surgery would involve and how their reconstructed breast or mastectomy scar might look following surgery. Shared decision-making between patient and clinician can mitigate this fear and provide women with a sense of autonomy over their health decisions. Provision of visual depictions of surgical outcomes was not routinely provided to those interviewed but was expressed as important to help women manage surgical expectations. Therefore our findings support the multi-modal presentation of diagnostic and treatment information to support decision-making. Likewise, women reported feeling unsupported in their decision not to undergo breast reconstruction, suggesting a need to develop resources to provide women with positive discussions about ‘going flat’.

Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase.

During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.

Enhancing the open-circuit voltage in narrow-bandgap CuInSe2 solar cells via local contact passivation with Al2O3

A fabrication technique using Al2O3-passivated local contacts was employed to produce narrow-bandgap CuInSe2 (CISe) photoabsorbers, which are well-suited as bottom cell materials in tandem devices. However, the performances of CISe cells with narrow bandgaps are impeded by the recombination of charge carriers, which reduces the open-circuit voltage (VOC). To overcome this limitation, an additional Al2O3 passivation layer was added to CISe solar cells. This enhanced the VOC while maintaining a spectral response of up to 1.0 eV, thereby boosting the photovoltaic conversion efficiency of the devices. Further, the Al2O3 passivation layer within the CISe absorber effectively mitigated the recombination of charge carriers, resulting in a substantial improvement in efficiency. Specifically, the Al2O3-passivated local contact hindered the charge-carrier recombination at the rear contact, leading to a marked increase in the VOC. Consequently, the overall photovoltaic conversion efficiency increased significantly from 10.4 % to 13 %. These results are expected to greatly further the development of CISe solar cells and achieve remarkable photovoltaic conversion efficiencies.

A superhydrophobic wearable rubber band with a synergistic dual conductive layer for monitoring human motions

Wearable and flexible electronics attract increasing attention due to their outstanding stretchability and human friendliness, while the significant parameters to evaluate sensor performance such as sensitivity and working range are the main challenge for better sensor design. Herein, an ultrasensitive and flexible strain sensor with synergistic dual conductive layers was successfully fabricated by designing a silk wrinkles structure to largely broaden the stretchable range for the conductive layer. The organic conductive polypyrrole and silver nanowire are combined to construct the conductive layer with high conductivity and better stretchability. The as-obtained sensor possesses a wide working range (0–126 %) and high sensitivity (a GF of 15710.75 at a strain of 120 %-126 %). The additional SiO2 layer is applied to the sensor to achieve a contact angle of 143.4°, which is close to super-hydrophobicity. The resultant sensor also possesses fast response and recovery speed due to the elastomeric rubber band substrate with the response and recovery time of 105 and 101 ms respectively, and still keeps high stability over 5000 cyclic stretching and releasing tests. The sensor is promising to be used in wearable applications potentially.

INVESTIGATING L2-L1 TRANSFER AMONG BILINGUAL STUDENTS IN THE REPUBLIC OF NORTH MACEDONIA

Language constitutes an indispensable facet of human existence It facilitates communication and perspective-sharing while allowing us to mould it to fit our unique needs. Foreign language proficiency adds an additional layer of such skills. Thus, people have been motivated to learn another language since time immemorial. As cultures and languages have intertwined and exposure to multimedia in different languages has grown, the number of bilinguals has grown, and with that, the opportunities for language transfer, as well. Language learners in North Macedonia, especially the ones who learn English have been becoming increasingly more proficient. This progress has been so substantial that educators have observed cases of influence from the second language (L2) to the first language (L1). Educators have noted various instances where this influence occurs such as: capitalizing certain words that aren’t meant to be capitalized in Macedonian, using English abbreviations incorrectly, mixing collocations, and atypical code-switching. Using observation during non–English classes and breaks, this small-scale study aimed to qualitatively investigate and interpret the L2-L1 transfer patterns in primary school students, and discuss them in a wider context using additional electronic resources. The results highlighted the numerous instances of such interlanguage influence i.e. mistakes that students make when using their mother tongue due to English.

The Parasporal Body of Bacillus thuringiensis subsp. israelensis: A Unique Phage Capsid-Associated Prokaryotic Insecticidal Organelle.

The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti's pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology.

High-density Fan-Out Chip on Substrate using M-Series (TM) and Adaptive Patterning® Technology

As silicon technology nodes advance and wafer costs increase, there is a strong incentive to shrink die size and concurrently, die pad pitch. This increased density typically drives higher cost advanced substrates with finer lines and spaces and potentially more layers. As a result of recent constraints on the substrate supply chain, this creates serious availability and cost concerns. A compelling solution is to use fan-out on substrate technology to increase the effective die pad pitch, allowing the use of simpler, lower layer count substrates. ASE used this single die Fan Out Chip on Substrate (FOCoS) approach in engineering and qualification builds starting in 2017 with a traditional fan-out solution. However, with increasing interconnect density, the conventional approach is limited in its ability to work with small bond pad pitches as the die shift during molding creates challenges for manufacturing. An oversized capture pad is often utilized to accommodate this shift and in one prominent case, an additional Cu layer is required. M-series’ use of Adaptive Patterning eliminates the need for oversized capture pads through a unique design-during-manufacturing process which precisely aligns the package interconnect layers to the actual location of each die after molding. This paper will examine the design and manufacturing of a FOCoS package using 4nm silicon through the cooperation of Deca and ASE. The design fans out the interface of a 12mm x 12mm die with 6,000 IO using a single RDL layer and 5 µm line and space. The die has a core pitch of 150µm with an average periphery pitch of 90µm. The resultant M-series flip-chip bumps have a core pitch of 150µm over 75% of the die area with a secondary pitch of 212µm encompassing the remaining perimeter of the die and the 30% body size growth provided by the fan-out region. The relaxed pitch provided by M-series fan-out opens the possibility to use the most cost-effective, readily available type of laminate substrate.

“I Felt like I Was Losing Every Day”

The covid-19 pandemic severely disrupted the education system in Canada from March 2020 throughout the 2020–21 school year. It also had disproportionate secondary effects on women in terms of unpaid care, economic loss, and poor mental health. This article explores the lived experience of women educators in the province of Alberta, drawing on interviews and focus groups with 39 educators. Findings indicate that the pandemic not only exacerbated the triple burden that women educators, in particular, bear but added additional layers of responsibility related to public health management, educating children at home, elder care responsibilities, and emotional labour. The essential role women educators fulfilled within the covid-19 response, at work and at home, cost them time, professional development opportunities, mental wellness, and the positive rewards that had drawn them to the educational field. Current concerns around educator burnout and retention may be mitigated by acting on the recommendations of women educators regarding the development of more equitable education systems and social policy.

The dangerous increasing support of Europol in national criminal investigations: An additional layer of complexity

Vice President for Promoting our European Way of Life Margaritis Schinas, stated at the time of the adoption of Europol’s amendment that “Europol is a true example of where EU action helps protect us all. Today’s agreement will give Europol the right tools and safeguard to support police forces in analysing big data to investigate crime and in developing pioneering methods to tackle cybercrime.” While some characterized the changes as an achievement for the adaptability and operational role of Europol, others argued that it undermines fundamental rights and weakens data protection. This paper analyses the amendments made to the Regulation and explores Europol’s increasing role of in national investigations and the associated dangers of it. The paper starts with a historical analysis of Europol’s legal framework and role in national criminal investigations, before diving into the core of the Regulation. After 2022, Europol supports Member States’ investigations in many ways. First, through the continuous retention of large and complex datasets, which was strongly criticized by NGOs and the EDPS. Second, through the transformation of Europol into the information hub and broker for the exchanges of data with private parties. Third, more indirectly, through Europol’s support of research and innovation projects, for national authorities to use and explore new technologies in their work. However, these amendments are not without dangers. The Regulation of 2022 pushes the boundaries of Europol’s competences further, by circumventing existing limits and questioning the legality of the operations. The stronger role of Europol lacks sufficient safeguards and efficient oversight. This is highly problematic considering the impact Europol may have on national investigations, and as a result on the situation of individuals.

Printing Parameter Optimization of Additive Manufactured PLA Using Taguchi Design of Experiment.

Three-dimensional printing (3DP), known as additive layer manufacturing (ALM), is a manufacturing process in which a three-dimensional structure is constructed by successive addition of deposited layers. Fused Deposition Modeling (FDM) has evolved as the most frequently utilized ALM process because of its cost-effectiveness and ease of operation. Nevertheless, layer adhesion, delamination, and quality of the finished product remain issues associated with the FDM process parameters. These issues need to be addressed in order to satisfy the requirements commonly imposed by the conventional manufacturing industry. This work is focused on the optimization of the FDM process and post-process parameters for Polylactic acid (PLA) samples in an effort to maximize their tensile strength. Infill density and pattern type, layer height, and print temperature are the process parameters, while annealing temperature is the post-process parameter considered for the investigation. Analysis based on the Taguchi L18 orthogonal array shows that the gyroid infill pattern and annealing cycle at 90 °C results in a maximum ultimate tensile strength (UTM) of 37.15 MPa. Furthermore, the regression model developed for the five variables under study was able to predict the UTS with an accuracy of more than 96%.

Biodistribution and function of coupled polymer-DNA origami nanostructures

Spatial control over the distribution of therapeutics is a highly desired feature, which could limit the side effects of many drugs. Here we describe a nanoscale agent, fabricated from a coupled polymer-DNA origami hybrid that exhibits stability in serum and slow diffusion through tissues, in a manner correlating with shape and aspect ratio. Coupling to fragments of polyethylene glycol (PEG) through polyamine electrostatic interactions resulted in marked stability of the agents in-vivo, with > 90% of the agents maintaining structural integrity 5 days following subcutaneous injection. An agent functionalized with aptamers specific for human tumor necrosis factor TNF-alpha, significantly abrogated the inflammatory response in a delayed-type hypersensitivity model in humanized TNF-alpha mice. These findings highlight polymer-DNA hybrid nanostructures as a programmable and pharmacologically viable update to mainstream technologies such as monoclonal antibodies, capable of exerting an additional layer of control across the spatial dimension of drug activity.

TMIC-57. CHARTING THE SPATIAL ARCHITECTURE OF GLIOBLASTOMA USING GBMAP: INSIGHTS FROM IN SITU SEQUENCING

Abstract Glioblastoma (GB), an aggressive brain malignancy, is associated with poor prognosis and quality of life. Single-cell RNA sequencing has helped unravel the complexity of GB cell states. However, to comprehensively unravel the pathobiology of GB, it is essential to consider the spatial context in which these cells reside within the tumor microenvironment (TME). In this study, we aimed to understand the spatial architecture of GB tumors and its implications on cellular and molecular heterogeneity. To achieve this, we developed GBmap, a comprehensive resource that harmonizes 26 datasets comprising 239 patients and over 1.1 million cells. Leveraging computational advancements, we utilized GBmap to create a GB-specific panel for charting the spatial architecture of tumors using an in situ sequencing (ISS)-based platform (HybISS) in an independent cohort of 13 tumors. We curated a panel of 209 genes based on the GBmap, capturing cell phenotypic features, intra- and inter-tissue heterogeneity, and in silico L-R analysis predictions. The panel includes markers for specific cellular processes such as inflammation, extracellular matrix remodeling, hypoxia, and angiogenesis, providing an additional layer of functional information. Our optimized pipeline incorporates a new method for ISS-based transcriptomics data (direct RNA targeting), which enhances sensitivity and tolerance for tissue specimens of poorer RNA quality. This new approach enables improved cell type/state resolution and classification confidence. Using this advanced approach, we observed heterogeneous cell-cell colocalization patterns that vary among patients, revealing spatiotemporal distributions of specific cancer phenotypes and distinct co-localization patterns with neighboring cells in the TME. Our findings showcase the unprecedented spatial and cellular resolution achieved, surpassing previous GB studies. Our study sheds light on the spatial architecture of GB tumors, unveiling previously unexplored cellular and molecular interactions. GBmap and our optimized pipeline offer a powerful resource for researchers to investigate GB heterogeneity, generate hypotheses, and contribute to advancing GB research.

Understanding the key risks in circular construction projects: from systematic review to conceptual framework

Purpose Circular construction offers sustainable solutions and opportunities to disentangle a project’s life cycle, including demolition, deconstruction and repurposing of architectural, civil engineering and infrastructure projects from the extraction of natural resources and their wasteful usage. However, it introduces additional layers of novel risks and uncertainties in the delivery of projects. The purpose of this study is to review the relevant literature to discover, classify and theorize the critical risk factors for circular construction projects. Design/methodology/approach The paper conducted a systematic literature review to investigate the risks of circular construction projects. It deployed a multistage approach, including literature search and assessment, metadata extraction, citation frequency analysis, Pareto analysis and total interpretive structural modeling. Findings Sixty-eight critical risk factors were identified and categorized into nine broad taxonomies: material risks, organizational risks, supply chain risks, technological risks, financial risks, design risks, health and safety risks, regulatory risks and stakeholder risks. Using the Pareto analysis, a conceptual map of 47 key critical risk factors was generated for circular construction projects. A hierarchical model was further developed to hypothesize the multiple possible connections and interdependencies of the taxonomies, leading to chain reactions and push effects of the key risks impacting circular construction projects. Originality/value This study constitutes the first systematic review of the literature, consolidating and theorizing the chain reactions of the critical risk factors for circular construction projects. Thus, it provides a better understanding of risks in circular construction projects.

Estimating genome-wide DNA methylation heterogeneity with methylation patterns

BackgroundIn a heterogeneous population of cells, individual cells can behave differently and respond variably to the environment. This cellular diversity can be assessed by measuring DNA methylation patterns. The loci with variable methylation patterns are informative of cellular heterogeneity and may serve as biomarkers of diseases and developmental progression. Cell-to-cell methylation heterogeneity can be evaluated through single-cell methylomes or computational techniques for pooled cells. However, the feasibility and performance of these approaches to precisely estimate methylation heterogeneity require further assessment.ResultsHere, we proposed model-based methods adopted from a mathematical framework originally from biodiversity, to estimate genome-wide DNA methylation heterogeneity. We evaluated the performance of our models and the existing methods with feature comparison, and tested on both synthetic datasets and real data. Overall, our methods have demonstrated advantages over others because of their better correlation with the actual heterogeneity. We also demonstrated that methylation heterogeneity offers an additional layer of biological information distinct from the conventional methylation level. In the case studies, we showed that distinct profiles of methylation heterogeneity in CG and non-CG methylation can predict the regulatory roles between genomic elements in Arabidopsis. This opens up a new direction for plant epigenomics. Finally, we demonstrated that our score might be able to identify loci in human cancer samples as putative biomarkers for early cancer detection.ConclusionsWe adopted the mathematical framework from biodiversity into three model-based methods for analyzing genome-wide DNA methylation heterogeneity to monitor cellular heterogeneity. Our methods, namely MeH, have been implemented, evaluated with existing methods, and are open to the research community.

Changing turn-over rates regulate abundance of tryptophan, GS biosynthesis, IAA transport and photosynthesis proteins in Arabidopsis growth defense transitions

BackgroundShifts in dynamic equilibria of the abundance of cellular molecules in plant-pathogen interactions need further exploration. We induced PTI in optimally growing Arabidopsis thaliana seedlings for 16 h, returning them to growth conditions for another 16 h.MethodsTurn-over and abundance of 99 flg22 responding proteins were measured chronologically using a stable heavy nitrogen isotope partial labeling strategy and targeted liquid chromatography coupled to mass spectrometry (PRM LC–MS). These experiments were complemented by measurements of mRNA and phytohormone levels.ResultsChanges in synthesis and degradation rate constants (Ks and Kd) regulated tryptophane and glucosinolate, IAA transport, and photosynthesis-associated protein (PAP) homeostasis in growth/PTI transitions independently of mRNA levels. Ks values increased after elicitation while protein and mRNA levels became uncorrelated. mRNA returned to pre-elicitation levels, yet protein abundance remained at PTI levels even 16 h after media exchange, indicating protein levels were robust and unresponsive to transition back to growth. The abundance of 23 PAPs including FERREDOXIN-NADP( +)-OXIDOREDUCTASE (FNR1) decreased 16 h after PAMP exposure, their depletion was nearly abolished in the myc234 mutant. FNR1 Kd increased as mRNA levels decreased early in PTI, its Ks decreased in prolonged PTI. FNR1 Kd was lower in myc234, mRNA levels decreased as in wild type.ConclusionsProtein Kd and Ks values change in response to flg22 exposure and constitute an additional layer of protein abundance regulation in growth defense transitions next to changes in mRNA levels. Our results suggest photosystem remodeling in PTI to direct electron flow away from the photosynthetic carbon reaction towards ROS production as an active defense mechanism controlled post-transcriptionally and by MYC2 and homologs. Target proteins accumulated later and PAP and auxin/IAA depletion was repressed in myc234 indicating a positive effect of the transcription factors in the establishment of PTI.

Regulatory roles of the N6-methyladenosine demethylase FTO in cardiac remodeling

Abstract Background The most prevalent internal RNA modification, N6-methyladenosine (m6A), serves as an additional layer of transcriptional and translational regulation. Methyl transferases (like METTL3) and demethylases (e.g., FTO) are the enzymes that catalyze m6A methylations. Our result showed that loss of FTO causes maladaptive cardiac remodeling in rodent hearts, which is consistent with other studies that emphasized the importance of FTO-dependent m6A RNA methylation in maintaining cardiac homeostasis, ventricular hypertrophy, and heart failure. Despite the fact that FTO is essential for stress-induced cardiac hypertrophy, its underlying functional mechanisms in regulating maladaptive cardiac remodeling are not yet known. Purpose In this study, we showed the necessity of FTO dependent m6A methylation in coordinating the molecular and signaling events that are engaged in cardiac hypertrophy, and further identified the detrimental signaling mechanisms that are regulated by FTO during pathological remodeling. Methods Firstly, the control and FTO-cKO (cardiomyocyte specific FTO knockout) group mice were subjected to sham and TAC (transverse aortic constriction) operation. Echocardiography was performed 1 week post-TAC. Secondly, m6A RNA immunoprecipitation (meRIP) sequencing were performed from the isolated mice heart tissue. Additionally, the effect of apoptosis and mTORC1 upon FTO depletion were analysed in both rodents and iPS-CMs. In vitro the interrelation between FTO demethylase, mTORC1 and autophagy in the regulation of cardiac apoptosis were identified by 1. inhibiting mTORC1 pathway with specific inhibitors, and by 2. inhibiting 'late autophagy' in FTO silenced iPS-CMs. Results In contrast to control mice, which exhibit concentric hypertrophy at the beginning of induced pressure overload (PO), echo analysis of one week post-TAC revealed that FTOcKO mice develop a DCM-like phenotype. Further, MeRIP-seq data showed that there is deregulation in the m6A methylation with FTO depletion. Around 114 differently hypermethylated transcripts and 58 differentially hypomethylated transcripts were discovered in FTOcKO sham as compared to Control sham; these transcripts are involved in the control of apoptosis, the mTORC1 signaling pathway, lysosomal biogenesis, and autophagy. In both rodents and iPS-CMs, FTO ablation dramatically enhances cardiac apoptosis and the mTORC1 signaling pathway. Furthermore, inhibition of mTORC1 pathway attenuates cardiac apoptosis in siFTO-iPS-CMs. Moreover, autophagic flux analysis revealed that FTO depletion cause autophagic flux impairment and exacerbate cardiac apoptosis. However, the autophagic flux is ameliorated with mTORC1 inhibition in late-autophagy inhibited siFTO-iPS-CMs. Conclusion Our data unravel the novel roles of FTO demethylase in the course of cardiac remodeling, which opens doors to therapeutic interventions in the treatment of cardiac hypertrophy and heart failure.

EMP3 sustains oncogenic EGFR/CDK2 signaling by restricting receptor degradation in glioblastoma

Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7 + late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display a transcriptomic signature consistent with reduced CDK2 activity, as well as increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, EMP3’s stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition.