51����

Dynamic regulation of protein tyrosine phosphorylation (pTyr) by kinases and phosphatases enables cells to sense and respond to environmental changes. The widely used chemical pervanadate induces the accumulation of pTyr in mammalian cell lines. This effect is primarily attributed to its inhibition of protein tyrosine phosphatases (PTPs), leading to the assertion that PTPs are master gatekeepers of intracellular pTyr homeostasis. Here, we used several approaches to reveal that pervanadate disrupted cellular redox homeostasis and directly activated tyrosine kinases of the SRC family through the oxidation of specific cysteine residues. Mass spectrometry and biophysical approaches showed that pervanadate-induced oxidation of cysteine-188 and cysteine-280 activated SRC by disrupting autoinhibitory intramolecular interactions between the catalytic domain and the SH2/SH3 domains and by impairing SH2 domain binding to phosphopeptides, including the regulatory carboxyl-terminal tail phosphotyrosine-530. Redox-sensitive cysteine residues were essential for SRC to promote the overgrowth of mouse fibroblasts. Our findings call for a reevaluation of pervanadate-based experiments and demonstrate that SRC cysteines control its oncogenic properties.

Infection triggers one of the most dramatic systemic responses in the body, and the coordinated activation and function of immune cells requires a dynamic regulation of transcriptomes and proteomes. This is achieved by RNA-binding proteins, which, together with RNA, form ribonucleoproteins. These proteins expand the information content of the genome and determine the lifespan, localization and function of RNA. Moreover, they control when, where and how much protein is produced. They can also mediate cell-autonomous immunity to foreign RNA and to misfolded self-RNAs and ensure the fidelity of the transcriptome by acting as RNA modifiers and chaperones to prevent RNA misfolding. These activities are integrated with gene expression programmes that are induced by the pathogen-sensing mechanisms of immune cells, which together activate, and later resolve, immune responses. Here, we review the activities of RNA-binding proteins in immune cells and discuss how perturbations of their function can result in immunodeficiency, autoimmunity and chronic inflammation.

Tendons are sparsely vascularized connective tissues that link muscles to bones, withstanding some of the highest mechanical stresses in the body. Mechanical overloading and tissue hypervascularity are implicated in tendinopathy, a common musculoskeletal disorder, yet their mechanistic roles remain unclear. Here, we identify hypoxia-inducible factor 1α (HIF1α) as not only a marker but also a driver of tendinopathy. Histological and multiomics evaluation of human tendinopathic samples revealed extensive extracellular matrix remodeling, including pathological collagen cross-linking coinciding with active hypoxic signaling. Hypothesizing a causal contribution of hypoxia signaling, we generated mice with tenocyte-targeted deletions of the von Hippel-Lindau () gene, which controls hypoxia signaling by regulating HIFα degradation. inactivation was sufficient to induce pathological hallmarks of tendinopathy, such as collagen matrix disorganization, cross-linking, altered mechanics, and neurovascular ingrowth. This phenotype was HIF1α dependent given that codeleting HIF1α rescued tendon morphology and mechanics. Moreover, deleting vascular endothelial growth factor A () alongside VHL effectively suppressed neovascularization but failed to rescue extracellular matrix abnormalities or restore mechanical function, emphasizing a direct role of HIF1α in driving tendon disease independently of angiogenesis. Mechanistically, we found that HIF1α activation was strain dependent in primary cultured human tendon cells and induced by mechanical overload in murine tendon explants. Furthermore, genetically removing α from tenocytes prevented aberrant tendon remodeling in response to chronic overload. These findings position HIF1α signaling as a central driver of tendinopathy that acts through a maladaptive tissue response to chronic overload, providing mechanistic insights that could be leveraged for therapeutic approaches.

Implantation of a human embryo into the endometrium is a crucial event in gestation, as it marks the initiation of a pregnancy and is prone to high failure rates. We have limited understanding of these stages because of the inaccessibility of implanting embryos and the lack of suitable model systems. Here, we establish an in vitro model that recapitulates the luminal, glandular, and stromal compartments of the superficial layer of receptive human endometrium. Human embryos and blastoids implant into the endometrial model, achieving post-implantation hallmarks including advanced trophoblast structures that underlie early events in placental development. Single-cell RNA sequencing of the embryo-endometrial interface at day 14 uncovers predicted molecular interactions between conceptus and endometrium. Disrupting signaling interactions between extravillous trophoblast and endometrial stromal cells caused defects in trophoblast outgrowth, demonstrating the importance of crosstalk processes to sustain embryogenesis. This platform opens the opportunity to investigate early stages of human embryo implantation.

Antibody feedback in germinal center (GC) responses plays a key role in shaping the affinity, specificity, and longevity of humoral immunity. Beyond neutralizing pathogens, antibodies influence B cell selection by modulating antigen availability and masking dominant epitopes, thereby reshaping the competitive landscape for T follicular helper (Tfh) cell support. This review outlines the current understanding of how antibody feedback governs the selection stringency and clonal evolution of GC B cells, facilitates and promotes the emergence of epitope spread, and contributes to GC shutdown. We also examine how it supports the development of broadly neutralizing antibodies. Finally, we discuss how these insights are informing next-generation vaccine strategies-including immunogen design, prime-boost regimens, and adjuvant optimization-to guide affinity maturation toward specific epitopes and overcome feedback-driven constraints. Understanding antibody feedback not only reveals fundamental principles of adaptive immunity but also offers new avenues for rational vaccine design and therapeutic immune modulation.

Cancer development is associated with dysregulation of the translatome, and targeting canonical eukaryotic initiation and elongation factors can offer treatment avenues for various neoplasms. Emerging evidence indicates that dysregulated mRNA elongation, involving alterations in eEF2 activity and eIF5A expression, also contributes to tumour cell growth. In this study, we investigate whether targeting eIF5A with the inhibitor GC7 is a viable strategy to curtail aberrant cell growth. Our findings demonstrate that inhibiting elongation by reducing eIF5A activity induces feedback inhibition of initiation through eIF2α phosphorylation, decreasing ternary complex formation and shutting down bulk protein synthesis. Employing dynamic SILAC, we identify proteins impacted by reduced eIF5A activity, and show their decreased translation results from feedback inhibition to initiation or other processes downstream of eIF5A. Decreased eIF5A activity impairs mitochondrial function, which activates signalling through HRI to eIF2α phosphorylation, reducing cancer cell proliferation. These effects are reversed by treatment with the integrated stress response inhibitor, implying that the impact of GC7 on cancer cell proliferation is mediated via translation initiation rather than elongation inhibition. These data suggest that eIF5A inhibition could be used to target cancer cells that depend on mitochondrial function for their proliferation and survival.

During peri-implantation development, the pluripotent tissue of the early embryo undergoes profound cellular and biochemical reprogramming. These transformations are essential for subsequent development, yet how they are coordinated with the preservation of genome integrity remains poorly understood. Here, we uncover a telomere length checkpoint that is elicited by metabolic remodeling as mouse embryonic stem cells (ESCs) transition from the naive to formative pluripotent state. We show that the exit of naive pluripotency is marked by accelerated mitochondrial respiration and de novo lipogenesis, fueling lipid droplet accumulation required for tissue remodeling. Unexpectedly, these acute metabolic shifts trigger transient telomere shortening and activate ZSCAN4, a pluripotency-associated regulator of telomeres, followed by telomere re-elongation as cells adopt a more glycolytic metabolic profile. Our findings reveal a feedback mechanism in which metabolism-induced telomere stress engages ZSCAN4 as a protective response, thereby linking metabolic state to telomere homeostasis during early developmental progression.

Pluripotency, the ability to generate all body cell types, emerges in a disorganized embryonic cell mass. After implantation, these cells form a columnar epithelium and initiate lumenogenesis. During gastrulation, some undergo epithelial-to-mesenchymal transition to form the primitive streak (PS). The signals controlling these events in humans are largely unknown. Here, to study them, we developed a chemically defined 3D model where conventional pluripotent stem cells self-organize into a columnar epithelium with a lumen, from which PS-like cells emerge. We show that early TGFβ family inhibition prevents epithelial identity, also in murine 3D embryo models and in embryos. ZNF398 acts downstream of TGFβ1, activating the epithelial master regulator ESRP1 while repressing mesenchymal factors CDH2 and ZEB2. After epithelium formation, TGFβ1 stimulation is dispensable for its maintenance. However, treatment via ACTIVIN-a distinct TGFβ family ligand-induces PS efficiently. Thus, signalling of the TGFβ family dynamically governs pluripotent epiblast epithelial identity.

Hypoxia is both a physiological and pathological signal in cells. Changes in gene expression play a critical role in the cellular response to hypoxia, enabling cells to adapt to reduced oxygen availability. These changes are primarily mediated by the HIF family of transcription factors, however, other transcription factors such as NF-κB, are also activated by hypoxia. Although NF-κB is known to be activated by hypoxia, the extent to which NF-κB contributes to the hypoxic response remains poorly understood. Here, we analysed hypoxia-induced, NF-κB-dependent gene expression, to define the NF-κB-dependent hypoxic signature. Our analysis reveals that most genes downregulated by hypoxia require NF-κB for their repression. We show that although the NF-κB-mediated hypoxic response may vary between cell types, a core subset of hypoxia-inducible genes requires NF-κB across multiple cell backgrounds. We demonstrate that NF-κB is critical for reactive oxygen species (ROS) generation and regulation of genes involved in oxidative phosphorylation under hypoxia. This work highlights NF-κB's central role in the hypoxia response and offering new insights into gene expression regulation by hypoxia and NF-κB.

Mutations in KRAS, particularly at codon 12, are frequent in adenocarcinomas of the colon, lungs and pancreas, driving carcinogenesis by altering cell signalling and reprogramming metabolism. However, the specific mechanisms by which different KRAS G12 alleles initiate distinctive patterns of metabolic reprogramming are unclear. Using isogenic panels of colorectal cell lines harbouring the G12A, G12C, G12D and G12V heterozygous mutations and employing transcriptomics, metabolomics, and extensive biochemical validation, we characterise distinctive features of each allele. We demonstrate that cells harbouring the common G12D and G12V oncogenic mutations significantly alter glutamine metabolism and nitrogen recycling through FOXO1-mediated regulation compared to parental lines. Moreover, with a combination of small molecule inhibitors targeting glutamine and glutamate metabolism, we also identify a common vulnerability that eliminates mutant cells selectively. These results highlight a previously unreported mutant-specific effect of KRAS alleles on metabolism and signalling that could be potentially harnessed for cancer therapy.

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5-null mouse model, which in germinal vesicle (GV) stage oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5-null oocytes, including at some imprinting control regions. This provides evidence for a mechanism of epigenetic impairment in oocytes which could contribute to downstream misregulation of imprinted genes.

Paradoxical activation of wild type RAF by chemical RAF inhibitors (RAFi) is a well-understood 'on-target' biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that requires the translation initiation factor eIF2α. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2α-dependent Integrated Stress Response (ISR). Rather, RAFi exposure activated GCN2, an alternate eIF2α kinase, leading to eIF2α-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2α antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic 'bell-shaped' concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by a GCN2 kinase dead mutation. A M802A GCN2 gatekeeper mutant was activated at lower RAFi concentrations, demonstrating that RAFi bind directly to the GCN2 kinase domain; this is supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation is a previously unappreciated off-target effect that may modulate tumour cell responses.

CD8 T cells target infected or malignant cells via the production of pro-inflammatory cytokines and direct target cell killing. Members of the ZFP36-family of RNA-binding proteins, ZFP36 and ZFP36L1, regulate these functions in T cells via the regulation of mRNA stability and protein translation. We investigate the regulation of ZFP36 and ZFP36L1 expression using in vitro differentiated OT1 TCR transgenic memory-like T cells. We characterise the differential kinetics and sensitivity of ZFP36 and ZFP36L1 to antigen affinity and PMA versus ionomycin stimulation. By selectively inhibiting TCR-induced signalling pathways, we find that p38 MAPK, MEK1/2, and PKC contribute to inducing both ZFP36 and ZFP36L1 expression. By contrast, inhibition of calcineurin using cyclosporin A potently inhibits ZFP36L1 expression while increasing and prolonging ZFP36 expression. The Zfp36 promoter contains many binding sites for the transcription factors ELK-1/4 and few binding sites for NFAT, while the Zfp36l1 promoter contains many NFAT binding sites and few ELK1/4 binding sites. Our findings suggest that the regulation of divergent transcription factors enables calcineurin to act as a signalling node that mediates the differential regulation of ZFP36 and ZFP36L1 during T cell activation.

The germinal center (GC) reaction drives the production of high-affinity antibodies by iterative cycles of B cell somatic hypermutation, selection, and proliferation. How GC B cells undergo rapid cell division while maintaining genome stability is poorly understood. Here, we show that the RNA binding proteins ZFP36L1 and ZFP36L2 act downstream of antigen sensing and protect GC B cells from replication stress by controlling a cell cycle-related posttranscriptional regulon. They safeguard the successful completion of mitosis by balancing CDK1 and p21-mediated regulation of cell-cycle progression. In their absence, GC B cells are prone to arrest in the G-M phase and die by apoptosis, resulting in curtailed GC responses. DNA replication forks stalled at active replication initiation zones, causing replication stress and increased activity of the ATR-CHK1 DNA damage response. Thus, RNA binding proteins guide posttranscriptional gene regulation and maintain a functional G-M checkpoint in GC B cells.

The guanine-nucleotide exchange factor (GEF) P-Rex1 mediates G protein-coupled receptor (GPCR) signaling by activating the small GTPase Rac. We show here that P-Rex1 also controls GPCR trafficking. P-Rex1 inhibits the agonist-stimulated internalization of the GPCR S1PR1 independently of its Rac-GEF activity, through its PDZ, DEP, and inositol polyphosphate 4-phosphatase domains. P-Rex1 also limits the agonist-induced trafficking of CXCR4, PAR4, and GLP1R but does not control steady-state GPCR levels, nor the agonist-induced internalization of the receptor tyrosine kinases PDGFR and EGFR. P-Rex1 blocks the phosphorylation required for GPCR internalization. P-Rex1 binds G protein-coupled receptor kinase 2 (Grk2), both in vitro and in cells, but does not appear to regulate Grk2 activity. We propose that P-Rex1 limits the agonist-induced internalization of GPCRs through its interaction with Grk2 to maintain high levels of active GPCRs at the plasma membrane. Therefore, P-Rex1 plays a dual role in promoting GPCR responses by controlling GPCR trafficking through an adapter function as well as by mediating GPCR signaling through its Rac-GEF activity.

P-Rex1 is a guanine-nucleotide factor for the small GTPase Rac (Rac-GEF) that is known to mediate neutrophil migration and ROS production in response to the activation of GPCRs. These roles of P-Rex1 are assumed to require its activation of Rac.

DNA methylation was the earliest epigenetic mark discovered-it is essential for mammalian development and forms a molecular memory that can transcend generations, as in the phenomenon of genomic imprinting. Set against this long-term potential, methylation is dynamic across the life cycle, with genome-wide changes at germ-cell specification, gametogenesis, and preimplantation development accompanying major shifts in cell potency. With a tool kit of precision genetic reagents, the mouse has been a mainstay in developing mechanistic understanding of how methylation is targeted to the genome and in exploring its susceptibility to environmental factors, such as parental diet. The availability of genome sequence from many more species combined with the ability to profile methylation and other epigenetic marks in very small numbers of cells now provides rich epigenomic information from other mammals. This information has begun to reveal both similarities as well as surprising differences in the way in which methylation is patterned across the genome among mammals. Such knowledge will be critical in assessing the outcomes of interventions during assisted reproduction in human clinical practice and livestock production.

Changes in transcript abundance and isoforms, mediated by epigenetic and post-transcriptional mechanisms, are a hallmark of the development, activation, and effector functions of immune cells. How epigenetic and post-transcriptional processes are orchestrated to regulate transcription and pre-mRNA processing, and their interplay with metabolism, is emerging as important for immunity. DNA and histone modifications recruit RNA-binding proteins (RBPs) to mediate co-transcriptional RNA processing at specific chromatin loci. Simultaneously, RBPs influence the deposition of epigenetic modifications by regulating the expression of chromatin-modifying enzymes and enzymes that control the amounts of metabolites. These are used as substrates by chromatin-modifying enzymes and can influence RBP activity; thus, modulation of metabolic pathways represents a mechanism to regulate the epigenetic landscape and pre-mRNA processing. A body of work identifies emerging regulatory principles that address the interplay between epigenetics and RBPs in the nucleus, and of cytoplasmic post-transcriptional mechanisms that regulate metabolism and epigenetics. In this review, we focus on the interconnections between RBP-mediated processes, chromatin modifications, and metabolic pathways, highlighting the role that such circuits have in T- and B-lymphocytes, and in autoimmunity.

We investigated the roles of Rac guanine-nucleotide exchange factor (Rac-GEF) P-Rex1 in glucose homeostasis using Prex1 and catalytically inactive Prex1 mice. P-Rex1 maintains fasting blood glucose levels and insulin sensitivity through its Rac-GEF activity but limits glucose clearance independently of its catalytic activity, throughout aging. Prex1 mice on a high-fat diet are protected from diabetes. The increased glucose clearance in Prex1 mice may stem in part from constitutively enhanced hepatic glucose uptake. P-Rex1 controls Glut2 surface levels and mitochondrial morphology, membrane potential, and ATP production in hepatocytes, independently of its catalytic activity. The inverse agonist GRA2 showed that P-Rex1 suppresses glucose uptake and mitochondrial ATP production in hepatocytes through the orphan GPCR Gpr21. Cell fractionation showed that P-Rex1 controls Gpr21 trafficking, independently of its catalytic activity. We propose that P-Rex1 limits hepatocyte glucose uptake by retaining Gpr21 at the plasma membrane. These findings delineate new strategies for controlling glucose homeostasis.

Chinese hamster ovary (CHO) cells are the leading mammalian system for recombinant therapeutic protein production. However, optimizing transgene expression remains challenging due to the limited understanding of the regulatory mechanisms controlling gene expression in CHO cells. Towards overcoming this barrier, here we provide a systematic characterization of cis-regulatory elements in CHO cells. Using genome-wide STARR-seq, a high-throughput method for quantifying enhancer strength, we identified regions with enhancer activity in the CHO cell genome. By integrating these data with ATAC-seq and histone modification profiles, we were able to characterize the chromatin state of these regions. Our analysis revealed thousands of newly identified enhancer sequences. The most active sequences could drive transgene expression at levels similar to or higher than strong viral enhancers. Notably, half of the regions found to have enhancer activity were within inaccessible chromatin in their native context. We observed that accessible enhancers were primarily near to transcriptional start sites and associated with ubiquitously-expressed genes, whereas inaccessible enhancers were predominantly intergenic and associated with tissue-specific genes. Additionally, through a deep-learning-based approach ETS and YY1 transcription factor (TF) binding motifs were identified as key determinants of enhancer identity and strength. Disrupting YY1 binding motifs led to reduced enhancer activity, thereby highlighting the importance of YY1 as a transcriptional activator in CHO cells. Our study demonstrates the first comprehensive map of functionally-validated enhancers in CHO cells and generates new insights into gene regulation and the role of TFs in determining enhancer strength. This study helps to lay the foundation for strategic engineering of CHO cell transcriptional networks to achieve enhanced biopharmaceutical production.

Tertiary lymphoid structures (TLSs) arise in non-lymphoid tissues in response to persistent antigen stimulation and chronic inflammation. Spanning organs from lung and liver to meninges, skin, and beyond, TLSs range from loose aggregates of immune cells to fully mature structures containing functional germinal centers (GC). In this review, we provide a comprehensive overview of TLS formation, architecture, and function across diverse tissues, highlighting both shared features and tissue-specific adaptations. We then explore the clinical relevance of TLS in infections, autoimmunity, cancer, and allergy, emphasizing their dual roles in mediating protective immunity and driving pathology. Finally, we discuss emerging technologies that are transforming our ability to dissect TLSs at high resolution (including spatial multi-omics, advanced imaging, and digital pathology), enabling mechanism-guided strategies to modulate TLSs therapeutically. Framing TLSs through the lens of maturation and tissue context provides a foundation for interpreting their clinical associations and for enhancing or dismantling these niches according to need.

In the twenty years since extrafollicular B cell responses were originally described, much has been learned about B cell biology. With this progress, the term "extrafollicular" has expanded beyond its initial use to describe a variety of B cell processes, resulting in ambiguity over the term. Extrafollicular responses are often not identified by location, convoluting the criteria being used to define the pathway. Here, we discuss the current understanding of B cell responses as relevant to the current uses of the term "extrafollicular." In this context, we propose a framework to classify evolving concepts in B cell biology. The use of this framework moving forward is expected to help harmonize and clarify the discussion in the field.

The diversity of antibodies underpins robust immune responses. During the formation of the antibody repertoire in early bone marrow B-cells, random antibody heavy-chain proteins are generated from recombined VH, DH, and JH gene segments. Many are non-functional and are negatively selected. To understand this process in normal mice, we have undertaken an in-depth analysis of heavy-chain selection at this pre-B cell transition. We find independent selection acting on three regions of the complementarity-determining region 3 (CDR3) antigen-binding site, with particularly heavy counter-selection against certain productive VH/JH combinations. This led us to hypothesise that VH-replacement, where the VH gene segment in an existing VDJ combination is replaced, targets productive VDJ rearrangements that code for non-functional heavy chains. We detect VH-replacement recombination products that closely follow the pattern of selection of functional and non-functional VDJ rearrangements. This reveals a physiological role for VH-replacement in the developmental release of B-cells that are stalled by non-functional heavy-chains. This leads to re-modelling of the restricted early VDJ repertoire toward the use of other VH gene segments throughout the IgH locus.

E-cadherin downregulation is an epithelial-mesenchymal transition hallmark canonically attributed to transcriptional repression. Here we delineate a metabolite-driven endocytic route of E-cadherin downregulation in inflammation-associated colorectal cancer (CRC). Specifically, IP kinase-2 (IP6K2), a 5-diphosphoinositol pentakisphosphate (5-IP) synthase upregulated in patients with CRC, is activated via a ROS-Src phosphorylation axis elicited by dextran sulfate sodium (DSS), generating 5-IP around adherens junction (AJ) to promote E-cadherin endocytosis and the transcriptional activities of β-catenin. Mechanistically, 5-IP inhibits inositol 5-phosphatases such as OCRL to promote PI(4,5)P-mediated endocytic adaptor recruitment. Depleting 5-IP or overexpressing a 5-IP binding-deficient OCRL mutant confers resistance to DSS-elicited AJ disruption. Intestinal epithelium-specific IP6K2 deletion attenuates DSS-induced colitis/CRC, whereas an IP6K2 isoform-selective inhibitor protects wild-type but not IP6K2 mice against DSS insult. Thus, 5-IP is an oncometabolite whose stimulus-dependent synthesis relieves a PI(4,5)P dephosphorylation-based endocytic checkpoint, leading to AJ disassembly and protumorigenic β-catenin activation. Targeting IP6K2 could strengthen intestinal epithelial barrier against inflammation and cancer.