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Journal of Clinical Biochemistry and Nutrition is a bi-monthly journal, currently published from Japan by the Society for free Radical Research. It publishes six issues in a year. The study examines the article published in the Journal of Clinical Biochemistry and Nutrition from 2007 to 2017. This paper aimed to assess growth pattern of research output, authorship pattern, degree of collaboration, ranking of authors based on productivity and h-index, most cited references, type of items produced, keyword wise distribution, most productive countries and most productive institutions, distributions of research output and also estimate the future growth of publications using straight line equation. There are 813 articles were published during 2007-2017(11 years). The highest number of 88 articles were published in 2009 and lowest number of 63 articles published in 2007. Majority of the contributions are more than five authors. There exists a higher level of collaborations between the authors. Naito Y is the most productive author ranked in first position and Suzuki H, Yoshikawa T and Watanabe K are each of them having h-index score 9 who are placed in the first rank. this study also reveals the highly cited papers in Journal of Clinical Biochemistry and Nutrition. Most of the research outputs in the journal are articles. 'Induced' is the keyword which is mostly occurred in the journal. Nearly one-third of the articles were published in Japan. It is known that different institutions were involved in the publication of articles, from these top most productive institutions are listed. It is estimated that research output of the source journal may take slightly increasing future. Keywords: Bibliometric, Clinical Biochemistry, Nutrition, Journal of Clinical Biochemistry and Nutrition, Quantitative analysis, Growth of publication, Citation, Authorship pattern, 1. INTRODUCTION Bibliometrics is a well-established research tool used by the librarians, teachers and information scientists to indicate the relationship between the cited and citing documents. The study is conducted [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.dsr.2005.12.001 Byline: Renato R. Neto (a), George A. Wolff (a), David S.M. Billett (b), Karen L. Mackenzie (a), Anu Thompson (a) Keywords: Porcupine abyssal plain; Holothurian; Lipid; Food supply; Temporal variability and response Abstract: The biochemical response of three species of deep-sea holothurian (Oneirophanta mutabilis, Pseudostichopus villosus, and Psychropotes longicauda) to temporal variation in food supply at the Porcupine Abyssal Plain (PAP; [approximately equal to]4850m water depth in the NE Atlantic) was studied over a period of 22 months. Lipid contents of P. longicauda showed a strong positive correlation with the contents of lipids in the surficial sediments (0-5mm; Spearman rank correlation, R.sub.s=1.0, P Author Affiliation: (a) Department of Earth and Ocean Sciences, University of Liverpool, 4 Brownlow Street, Liverpool, L69 3GP, UK (b) National Oceanography Centre, DEEPSEAS Benthic Biology Group, Southampton, SO14 3ZH, UK Article History: Received 16 March 2005; Revised 23 November 2005; Accepted 14 December 2005

A new mutual information (MI)-based feature-selection method to solve the so-called large p and small n problem experienced in a microarray gene expression-based data is presented. First, a grid-based feature clustering algorithm is introduced to eliminate redundant features. A huge gene set is then greatly reduced in a very efficient way. As a result, the computational efficiency of the whole feature-selection process is substantially enhanced. Second, MI is directly estimated using quadratic MI together with Parzen window density estimators. This approach is able to deliver reliable results even when only a small pattern set is available. Also, a new MI-based criterion is proposed to avoid the highly redundant selection results in a systematic way. At last, attributed to the direct estimation of MI, the appropriate selected feature subsets can be reasonably determined. Index Terms--Cancer diagnosis, feature selection, grid-based redundancy elimination, microarray gene expression data, quadratic mutual information (QMI).

Post-embryonic diapause in copepods is an adaptation that allows species in the copepod family Calanidae to thrive in high-latitude environments by transforming a short spring phytoplankton bloom into large numbers of lipid-rich individuals capable of surviving a long period of starvation. The copepods, with their high-energy lipid reservoirs, are a critical food source for higher trophic levels, making the Calanidae a key component of high-latitude marine ecosystems. The physiological ecology of the developmental program remains poorly understood. However, new studies using high-throughput RNA sequencing approaches are giving detailed access to physiological status by generating gene expression profiles for both field-collected and laboratory-incubated individuals. These are beginning to characterize the diapause phenotype, elucidate the transcriptional and physiological progression through the diapause program, and illustrate the effects of organism-environment interactions. This paper reviews gene expression profiling studies on the life cycle and diapause program of Neocalanus flemingeri Miller (1988) that were conducted as part of a long-term observation program in the north-em Gulf of Alaska. It summarizes recent findings and relates them to the ecology of this species and to that of other calanids., Introduction Post-embryonic diapause is widespread in the high-latitude copepods in the families Calanidae and Eucalanidae. These copepods transform a short phytoplankton bloom into a lipid-rich food source that persists beyond [...]

This paper analysed Sickle cell anaemia literature on Nigeria listed in PubMed to understand the research performance of medical researchers on sickle cell anaemia literature on Nigeria. Data covering the period of 2006-2016 were collected from the PubMed, a citation database developed by the National Centre for Biotechnology Information (NCBI).The search strategy used was ((sickle cell anaemia) OR (sickle cell anemia)) AND Nigeria AND (('2006/01/01'[PDat]: '2016/12/31'[PDat])), bearing in mind variance in spelling (anaemia and anemia), which retrieved 326 sickle cell literature on Nigeria between 2006 and 2016. Article productivity increased between 2006 and 2010. However, there was a drastic drop in article publication between 2010 and 2011 as well as between 2013 and 2015. The degree of collaboration ranged between 0.85 and 1. University of Nigeria Teaching Hospital, Enugu, Nigeria, had the highest number of contributing authors. Authors published more in journals located in Nigeria, with Nigeria Journal of Clinical Practice being the most prolific publication outlet for authors publishing sickle cell anaemia literature on Nigeria. Keywords Bibliometric Analysis, Sickle Cell Anaemia, Nigeria, PubMed, Introduction Sickle cell anaemia, recognized as a major public health problem by the World Health Organization (WHO) (WHO, 2006) is a genetic blood disorder characterized by abnormal red blood cells [...]

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss., Introduction Disabling hearing loss affects approximately 6.1% of the global population (1). Most hearing loss is due to death of sensory hair cells of the inner ear. These mechanosensitive receptor [...]

Inherited retinal degenerations (IRDs) are characterized by the progressive loss of photoreceptors and represent one of the most prevalent causes of blindness among working-age populations. Cyclic nucleotide dysregulation is a common pathological feature linked to numerous forms of IRD, yet the precise mechanisms through which this contributes to photoreceptor death remain elusive. Here we demonstrate that cAMP induced upregulation of the dependence receptor neogenin in the retina. Neogenin levels were also elevated in both human and murine degenerating photoreceptors. We found that overexpressing neogenin in mouse photoreceptors was sufficient to induce cell death, whereas silencing neogenin in degenerating murine photoreceptors promoted survival, thus identifying a pro-death signal in IRDs. A possible treatment strategy is modeled whereby peptide neutralization of neogenin in Rd1, Rd10, and Rho P23H-knockin mice promotes rod and cone survival and rescues visual function as measured by light-evoked retinal ganglion cell recordings, scotopic/photopic electroretinogram recordings, and visual acuity tests. These results expose neogenin as a critical link between cAMP and photoreceptor death, and identify a druggable target for the treatment of retinal degeneration., Introduction In industrialized countries, a major cause of blindness is the progressive death and dysfunction of photoreceptors (1), most commonly due to age-related macular degeneration (AMD) and inherited retinal degenerations [...]

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-[kappa]B, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in [Fanca.sup.-/-] mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence., Introduction Hematopoiesis is a fine-tuned process in which a small pool of specialized cells, hematopoietic stem cells (HSCs), provide the BM and the bloodstream with undifferentiated and differentiated cells throughout [...]

Immunotherapy targeting programmed cell death-1 (PD-1) induces durable antitumor efficacy in many types of cancer. However, such clinical benefit is limited because of the insufficient reinvigoration of antitumor immunity with the drug alone; therefore, rational therapeutic combinations are required to improve its efficacy. In our preclinical study, we evaluated the antitumor effect of U3-1402, a human epidermal growth factor receptor 3-targeting (HER3-targeting) antibody-drug conjugate, and its potential synergism with PD-1 inhibition. Using a syngeneic mouse tumor model that is refractory to anti-PD-1 therapy, we found that treatment with U3-1402 exhibited an obvious antitumor effect via direct lysis of tumor cells. Disruption of tumor cells by U3-1402 enhanced the infiltration of innate and adaptive immune cells. Chemotherapy with exatecan derivative (Dxd, the drug payload of U3-1402) revealed that the enhanced antitumor immunity produced by U3- 1402 was associated with the induction of alarmins, including high-mobility group box-1 (HMGB-1), via tumor-specific cytotoxicity. Notably, U3-1402 significantly sensitized the tumor to PD-1 blockade, as a combination of U3-1402 and the PD-1 inhibitor significantly enhanced antitumor immunity. Further, clinical analyses indicated that tumor-specific HER3 expression was frequently observed in patients with PD-1 inhibitor-resistant solid tumors. Overall, U3-1402 is a promising candidate as a partner of immunotherapy for such patients., Introduction Immune checkpoint inhibitor (ICI) therapy has emerged as a standard of care treatment for many types of cancer. The mainstay of such immunotherapy is the programmed cell death-1 (PD-1)/programmed [...]

The shrimp Pleoticus muelleri (Bale, 1888) is an important fishing product from the Southwest Atlantic waters which is frequently captured as a bycatch and discarded. Wastes and by-products of shrimp are an excellent source of protein and enzymes. This review highlights the biochemical and catalytic properties of proteases from shrimp. Previous studies on P. muelleri enzymes have found different activities of alkaline proteases ranging from 0.01 to 0.78 Abs/min/mg of protein. These changeful results might be explained by the great influence that several biological and experimental factors, such as molting and developmental stages, type of diet, and culture conditions, have on the enzyme function. Regarding acid protease, it has been demonstrated that they have activities between 0.3 and 0.4 Abs/min/mg of protein. Processing Argentine red shrimp waste could offer many higher value products without increasing wild catch volumes; therefore, it is essential to conduct further studies that allow the development of more value-added products derived from the practical usage of shrimp processing waste. KEY WORDS: shrimp, Pleoticus muelleri, aquaculture, by-products, cheese, fishery, proteases, wastes, INTRODUCTION Fish processing industries produce a large number of by-products as waste (viscera, heads, tails, skins, and shells) in all the stages of the fish production value chain. Disposal of [...]

As an essential mineral, magnesium plays a vital role in human biochemistry and general health. It is involved in over 300 biochemical reactions in the body. (1) Subsequently, a high [...]

During developmental angiogenesis, blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether, how, cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates cell death via both apoptosis and necroptosis. Here, we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal retina angiogenesis. EC-specific Casp-8-KO pups (Casp-[8.sup.ECKO]) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting, and migration independently of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 MAPK downstream of receptor-interacting serine/threonine protein kinase 3 (RIPK3) and destabilization of vascular endothelial cadherin (VE-cadherin) at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR) resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-[8.sup.ECKO] pups. Taking these data together, we show that Casp-8 acts in a cell death-independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP., Introduction Functional blood vessels are of vital importance. Impaired vessel formation contributes to many pathological situations, including ischemic and inflammatory disorders. Ischemic retinopathies are the main causes of severe visual [...]

Histone H3K27 demethylase JMJD3 plays a critical role in gene expression and T cell differentiation. However, the role and mechanisms of JMJD3 in T cell trafficking remain poorly understood. Here, we show that JMJD3 deficiency in [CD4.sup.+] T cells resulted in an accumulation of T cells in the thymus and reduction of T cell number in the secondary lymphoid organs. We identified PDLIM4 as a significantly downregulated target gene in JMJD3- deficient [CD4.sup.+] T cells by gene profiling and ChIP-Seq analyses. We further showed that PDLIM4 functioned as an adaptor protein to interact with sphingosine-1 phosphate receptor 1 (S1P1) and filamentous actin (F-actin), thus serving as a key regulator of T cell trafficking. Mechanistically, JMJD3 bound to the promoter and gene-body regions of the Pdlim4 gene and regulated its expression by interacting with zinc finger transcription factor KLF2. Our findings have identified Pdlim4 as a JMJD3 target gene that affects T cell trafficking by cooperating with S1P1 and have provided insights into the molecular mechanisms by which JMJD3 regulates genes involved in T cell trafficking., Introduction T cell development in the thymus is a multistep process. Early thymic progenitor cells (TPCs) differentiate into T cell receptor-expressing (TCR-expressing) [CD4.sup.+][CD8.sup.+] double-positive (DP) thymocytes in the cortex and [...]

The prognostic value of immune cell infiltration within the tumor microenvironment (TME) has been extensively investigated via histological and genomic approaches. Based on the positive prognostic value of T cell infiltration, Immunoscore has been developed and validated for predicting risk of recurrence for colorectal cancer (CRC). Also, association between a consensus T helper 1 (Th-1) immune response and favorable clinical outcomes has been observed across multiple cancer types. Here, we reanalyzed public genomic data sets from The Cancer Genome Atlas (TCGA) and NCBI Gene Expression Omnibus (NCBI-GEO) and performed multispectral immunohistochemistry (IHC) on a cohort of colorectal tumors. We identified and characterized a risk group, representing approximately 10% of CRC patients, with high intratumoral [CD8.sup.+] T cell infiltration, but poor prognosis. These tumors included both microsatellite instable (MSI) and stable (MSS) phenotypes and had a high density of tumor-associated macrophages (TAMs) that expressed CD274 (programmed death-ligand 1 [PD-L1]), TGF-[beta] activation, and an immune overdrive signature characterized by the overexpression of immune response and checkpoint genes. Our findings illustrate that CRC patients may have poor prognosis despite high [CD8.sup.+] T cell infiltration and provide CD274 as a simple biomarker for identifying these patients., Introduction Since the seminal report on association between infiltrating cytotoxic and memory T cells with decreased lymphatic invasion and improved patient outcome in colorectal cancer (CRC) (1), the prognostic value [...]

Cancer immunotherapy restores or enhances the effector function of CD8.sup.+ T cells in the tumour microenvironment.sup.1,2. CD8.sup.+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways.sup.3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide.sup.5,6. Although it has been investigated in vitro.sup.7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios.sup.9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8.sup.+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFN[gamma]) released from CD8.sup.+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system x.sub.c.sup.-, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system x.sub.c.sup.- was negatively associated, in cancer patients, with CD8.sup.+ T cell signature, IFN[gamma] expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFN[gamma] and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach. Interferon-[gamma] induces ferroptotic cell death in tumours by suppressing cystine uptake and promoting lipid peroxidation., Author(s): Weimin Wang [sup.1] [sup.2] , Michael Green [sup.2] [sup.3] , Jae Eun Choi [sup.2] [sup.4] [sup.5] , Miguel Gijón [sup.6] , Paul D. Kennedy [sup.6] , Jeffrey K. Johnson [...]

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments.sup.1,2. These biomolecular condensates are formed from processes that include liquid-liquid phase separation. The multivalent interactions necessary for liquid-liquid phase separation have been extensively studied in vitro.sup.1,3. However, the regulation of this process in vivo is poorly understood. Here we identify an in vivo regulator of liquid-liquid phase separation through a genetic screen targeting factors required for Arabidopsis RNA-binding protein FCA function. FCA contains prion-like domains that phase-separate in vitro, and exhibits behaviour in vivo that is consistent with phase separation. The mutant screen identified a functional requirement for FLL2, a coiled-coil protein, in the formation of FCA nuclear bodies. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome.sup.3,4. FLL2 was required to promote this proximal polyadenylation, but not the binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3'-end processing machinery. These 3' RNA-processing components colocalized with FCA in the nuclear bodies in vivo, which indicates that FCA nuclear bodies compartmentalize 3'-end processing factors to enhance polyadenylation at specific sites. Our findings show that coiled-coil proteins can promote liquid-liquid phase separation, which expands our understanding of the principles that govern the in vivo dynamics of liquid-like bodies. A genetic screen for factors required by the Arabidopsis RNA-binding protein FCA identifies FLL2 as necessary in the formation of FCA nuclear bodies, and thus a role for FLL2 in liquid-liquid phase separation., Author(s): Xiaofeng Fang [sup.1] , Liang Wang [sup.2] , Ryo Ishikawa [sup.1] [sup.5] , Yaoxi Li [sup.1] , Marc Fiedler [sup.3] , Fuquan Liu [sup.1] [sup.6] , Grant Calder [sup.1] [...]

[Na.sup.+]/[H.sup.+] exchanger isoform one (NHE1) is a mammalian plasma membrane protein that removes intracellular protons, thereby elevating intracellular pH ([pH.sub.i]). NHE1 uses the energy of allowing an extracellular sodium down its gradient into cells to remove one intracellular proton. The ubiquitous protein has several important physiological and pathological influences on mammalian cells as a result of its activity. The three-dimensional structure of human NHE1 (hNHE1) is not known. Here, we modeled NHE1 based on the structure of MjNhaP1 of Methanocaldoccocus jannaschii in combination with biochemical surface accessibility data. hNHE1 contained 12 transmembrane segments including a characteristic [Na.sup.+]/[H.sup.+] antiporter fold of two trans-membrane segments with a helix--extended region--helix conformation crossing each other within the membrane. Amino acids 363-410 mapped principally to the extracellular surface as an extracellular loop (EL5). A large preponderance of amino acids shown to be surface accessible by biochemical experiments mapped near to, or on, the extracellular surface. Docking of [Na.sup.+]/[H.sup.+] exchanger inhibitors to the extracellular surface suggested that inhibitor binding on an extracellular site is made up from several amino acids of different regions of the protein. The results present a novel testable, three-dimensional model illustrating NHE1 structure and accounting for experimental biochemical data. Key words: molecular modeling, MjNhaP1, [Na.sup.+]/[H.sup.+] exchanger, NhaA, NHE1. L'echangeur [Na.sup.+]/[H.sup.+] NHE1 est une proteine de la membrane plasmique des mammiferes qui enleve les protons intracellulaires, elevant ainsi le pH intracellulaire ([pH.sub.i]). NHE1 utilise l'energie generee par l'entree spontanee dans les cellules d'un sodium extracellulaire par gradient pour faire sortir un proton intracellulaire. Cette proteine ubiquiste exerce une influence importante sur les plans physiologiques et pathologiques dans les cellules de mammiferes consequemment a son activite. La structure tridimensionnelle de NHE1 humain (hNHE1) n'est pas connue. Les auteurs ont modelise ici NHE1 a partir de la structure de MjNhaP1 de Methanocaldococcus jannaschii en combinaison avec les donnees biochimiques d'accessibilite de la surface. Le transporteur hNHE1 comprenait 12 segments transmembranaires dont un repli caracteristique de l'antiport [Na.sup.+]/[H.sup.+], formededeux segments transmembranaires dans une conformation helice--region etendue--helice qui se croisental'interieur de la membrane. Les acides amines 363-410 ont ete cartographies principalement a la surface extracellulaire en tant que boucle extracellulaire (EL5). Une forte preponderance d'acides amines accessibles a la surface selon des experiences biochimiques etait cartographiee a proximite ou sur la surface extracellulaire. L'amarrage d'inhibiteurs de l'echangeur [Na.sup.+]/[H.sup.+] a la surface extra-cellulaire suggerait que la liaison de l'inhibiteur sur un site extracellulaire implique plusieurs acides amines de differentes regions de la proteine. Les resultats presentent un nouveau modele tridimensionnel verifiable qui illustre la structure de NHE1 et explique les donnees biochimiques experimentales. [Traduit par la Redaction] Mots-cles : modelisation moleculaire, MjNhaP1, echangeur [Na.sup.+]/[H.sup.+], NhaA, NHE1., Introduction Membranes are key to cellular function. They separate cells from the extracellular environment and allow cells to maintain intracellular compartments of specific content. The plasma membrane of eukaryotic cells [...]

A living organism is a biochemically unique system, and its vital functions can be investigated using analytical measurements. Characteristic chromatographic or electrophoretic profiles of biologically active substances have been the objects of active biomedical research for more than 15 years because of the possibility of rapidly obtaining diagnostically important information. In this review, targeted and untargeted metabolomic studies performed by chromatography and capillary electrophoresis are considered., Author(s): L. A. Kartsova [sup.1] , S. A. Solov'eva [sup.1] Author Affiliations: (Aff1) 0000 0001 2289 6897, grid.15447.33, Institute of Chemistry, St. Petersburg State University, , 198504, St. Petersburg, Russia [...]

Allergic diseases have in common a dysfunctional epithelial barrier, which allows the penetration of allergens and microbes, leading to the release of type 2 cytokines that drive allergic inflammation. The accessibility of skin, compared with lung or gastrointestinal tissue, has facilitated detailed investigations into mechanisms underlying epithelial barrier dysfunction in atopic dermatitis (AD). This Review describes the formation of the skin barrier and analyzes the link between altered skin barrier formation and the pathogenesis of AD. The keratinocyte differentiation process is under tight regulation. During epidermal differentiation, keratinocytes sequentially switch gene expression programs, resulting in terminal differentiation and the formation of a mature stratum corneum, which is essential for the skin to prevent allergen or microbial invasion. Abnormalities in keratinocyte differentiation in AD skin result in hyperproliferation of the basal layer of epidermis, inhibition of markers of terminal differentiation, and barrier lipid abnormalities, compromising skin barrier and antimicrobial function. There is also compelling evidence for epithelial dysregulation in asthma, food allergy, eosinophilic esophagitis, and allergic rhinosinusitis. This Review examines current epithelial barrier repair strategies as an approach for allergy prevention or intervention., Allergic diseases such as atopic dermatitis (AD), food allergy (FA), asthma, and allergic rhinitis affect more than 30% of the population (1-3). These diseases have in common a dysfunctional epithelial [...]

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells.sup.1. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRP[alpha] expressed on myeloid cells.sup.2-5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRP[alpha] checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRP[alpha] binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer. QPCTL is a modifier of CD47-SIRP[alpha] binding and its blockade enhances macrophage- and neutrophil-mediated antibody dependent cellular cytotoxicity towards tumor cells., Author(s): Meike E. W. Logtenberg [sup.1] , J. H. Marco Jansen [sup.2] , Matthijs Raaben [sup.3] , Mireille Toebes [sup.1] , Katka Franke [sup.4] , Arianne M. Brandsma [sup.2] , [...]

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts. A bacterial family of cGAS/DncV-like nucleotidyltransferases synthesizes a diverse range of cyclic dinucleotide and trinucleotide compounds that are likely to modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts., Author(s): Aaron T. Whiteley [sup.1] [sup.2] , James B. Eaglesham [sup.1] [sup.2] , Carina C. de Oliveira Mann [sup.1] [sup.2] , Benjamin R. Morehouse [sup.1] [sup.2] , Brianna Lowey [sup.1] [...]

Preterm birth ( Key words: cyclooxygenase, cyclooxygenase inhibitors, preterm, labour, pregnancy, prostaglandins, tocolysis, myometrium, decidua, fetal membranes. La naissance prematuree ( Mots-cles : cyclooxygenase, inhibiteurs de la cyclooxygenase, premature, travail, grossesse, prostaglandines, tocolyse, myometre, deciduale, membranes fretales., Introduction Preterm birth ( Indeed, administration of the nonselective COX inhibitor indomethacin to women in preterm labour led to a dramatic cessation of uterine contractions (Zuckerman et al. 1974; Gyoiy [...]

Genetic variants at the PTPN2 locus, which encodes the tyrosine phosphatase PTPN2, cause reduced gene expression and are linked to rheumatoid arthritis (RA) and other autoimmune diseases. PTPN2 inhibits signaling through the T cell and cytokine receptors, and loss of PTPN2 promotes T cell expansion and CD4- and [CD8.sup.- ]driven autoimmunity. However, it remains unknown whether loss of PTPN2 in [FoxP3.sup.+] regulatory T cells (Tregs) plays a role in autoimmunity. Here we aimed to model human autoimmune-predisposing PTPN2 variants, the presence of which results in a partial loss of PTPN2 expression, in mouse models of RA. We identified that reduced expression of Ptpn2 enhanced the severity of autoimmune arthritis in the T cell-dependent SKG mouse model and demonstrated that this phenotype was mediated through a Treg-intrinsic mechanism. Mechanistically, we found that through dephosphorylation of STAT3, PTPN2 inhibits IL-6-driven pathogenic loss of FoxP3 after Tregs have acquired ROR[gamma]t expression, at a stage when chromatin accessibility for STAT3-targeted [IL-17.sup.-]associated transcription factors is maximized. We conclude that PTPN2 promotes FoxP3 stability in mouse [ROR[gamma]t.sup.+] Tregs and that loss of function of PTPN2 in Tregs contributes to the association between PTPN2 and autoimmunity., Introduction Rheumatoid arthritis (RA) is a chronic autoimmune, systemic inflammatory disorder that primarily affects diarthrodial joints (1). To date, various genome-wide association studies have identified over 100 risk loci for [...]

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs. The transcription factor PU.1 is an essential regulator of the pro-fibrotic gene expression program in fibroblasts; PU.1 expression is upregulated in various fibrotic diseases, whereas inactivation of PU.1 induces regression of fibrosis in a number of organs., Author(s): Thomas Wohlfahrt [sup.1] , Simon Rauber [sup.1] , Steffen Uebe [sup.2] , Markus Luber [sup.1] , Alina Soare [sup.1] , Arif Ekici [sup.2] , Stefanie Weber [sup.1] , Alexandru-Emil [...]

Our understanding of metabolic networks is incomplete, and new enzymatic activities await discovery in well-studied organisms. Mass spectrometric measurement of cellular metabolites reveals compounds inside cells that are unexplained by current maps of metabolic reactions, and existing computational models are unable to account for all activities observed within cells. Additional large-scale genetic and biochemical approaches are required to elucidate metabolic gene function. We have used full-scan mass spectrometry metabolomics of polar small molecules to examine deletion mutants of candidate enzymes in the model yeast Saccharomyces cerevisiae. We report the identification of 25 genes whose deletion results in focal metabolic changes consistent with loss of enzymatic activity and describe the informatic approaches used to enrich for candidate enzymes from uncharacterized open reading frames. Triumphs and pitfalls of metabolic phenotyping screens are discussed, including estimates of the frequency of uncharacterized eukaryotic genes that affect metabolism and key issues to consider when searching for new enzymatic functions in other organisms.Key words: metabolomics, enzyme discovery, discovery metabolite profiling, hexosepyranosyl-citrulline.Notre comprehension des reseaux metaboliques est incomplete et de nouvelles activites enzymatiques tardent a etre decouvertes dans des organismes bien etudies. La mesure par spectrometrie de masse de metabolites cellulaires revele la presence de composes a l'interieur des cellules qui sont inexpliques par la cartographie actuelle des reactions metaboliques, et les modeles computationnels existants sont incapables d'expliquer toutes les activites observees a l'interieur des cellules. De nouvelles approches genetiques et biochimiques a grande echelle sont necessaires pour elucider la fonction des genes du metabolisme. Les auteurs ont utilise la metabolomique en spectrometrie de masse a balayage complet de petites molecules polaires afin d'examiner des mutants de deletion d'enzymes candidates chez la levure modele Saccharomyces cerevisiae. Ils rapportent l'identification de 25 genes dont la deletion donne lieu a des changements metaboliques focalises coherents avec la perte d'activite enzymatique, et decrivent les approches informatiques utilisees pour enrichir le groupe d'enzymes candidates a partie de cadres de lecture ouverts non caracterises. Les succes et les echecs des criblages du phenotypage metabolique sont discutes, dont les estimations de la frequence des genes eucaryotes non caracterises qui affectent le metabolisme, ainsi que les questions cles a considerer lors de la recherche de nouvelles fonctions enzymatiques chez d'autres organismes. [Traduit par la Redaction]Mots-cles : metabolomique, decouverte d'enzymes, profilage metabolique, hexosepyranosyl-citrulline., IntroductionUncharacterized enzymes remain to be discovered even in well-studied genomesMany gaps in the collective understanding of metabolism remain despite decades of work invested by the scientific community to both identify [...]

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP-RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.Key words: hematopoietic stem cells, leukemia stem cells, post-transcriptional regulation, RNA-binding proteins, RNA regulons.L'hematopoiese normale est soutenue par un equilibre soigneusement orchestre entre l'autorenouvelement des cellules souches hematopoietiques (CSH) et leur differenciation. L'importance fonctionnelle de cet axe est mise en evidence par la severite des phenotypes des maladies initiees par une fonction anormale des CSH, dont les syndromes myelodysplasiques et les cancers hematopoietiques. Des percees importantes dans la comprehension de la regulation transcriptionnelle des cellules hematopoietiques primitives ont ete realisees; toutefois, en comparaison, les composantes de la regulation post-transcriptionnelle qui pourraient affecter leur comportement demeurent peu explorees. Les joueurs cles de ce niveau de regulation comprennent les proteines de liaison de l'ARN qui executent un controle hautement coordonne de l'expression genique par la modulation des proprietes de l'ARN dont l'epissage, la polyadenylation, la localisation, la degradation ou la traduction. Avec l'identification recente de proteines de liaison de l'ARN qui jouent un role essentiel dans la regulation de la proliferation et des decisions cellulaires dans d'autres systemes, l'importance du controle post-transcriptionnel a l'echelle des cellules souches est de plus en plus reconnue. Les auteurs discutent ici de la comprehension actuelle de la regulation post-transcriptionnelle assuree par les proteines de liaison de l'ARN dans les CSH, de son implication dans l'hematopoiese normale, perturbee et maligne, de meme que des innovations technologiques les plus recentes visant a caracteriser le reseau ARN-proteines de liaison de l'ARN a l'echelle des systemes. De nouvelles donnees soulignent que le controle exerce par les proteines de liaison de l'ARN constitue une caracteristique negligee de l'hematopoiese primitive, dont une meilleure connaisance aurait d'importantes retombees cliniques. [Traduit par la Redaction]Mots-cles : cellules souches hematopoietiques, cellules souches leucemiques, regulation post-transcriptionnelle, proteines de liaison de l'ARN, regulons d'ARN., IntroductionThe machinery underlying stem cells' capacity to orchestrate their self-maintenance and differentiation can be represented as integrated circuits that are genetically programmed. As demonstrated by the capacity to reprogram somatic [...]

Mammalian gene expression is inherently stochastic.sup.1,2, and results in discrete bursts of RNA molecules that are synthesized from each allele.sup.3-7. Although transcription is known to be regulated by promoters and enhancers, it is unclear how cis-regulatory sequences encode transcriptional burst kinetics. Characterization of transcriptional bursting, including the burst size and frequency, has mainly relied on live-cell.sup.4,6,8 or single-molecule RNA fluorescence in situ hybridization.sup.3,5,8,9 recordings of selected loci. Here we determine transcriptome-wide burst frequencies and sizes for endogenous mouse and human genes using allele-sensitive single-cell RNA sequencing. We show that core promoter elements affect burst size and uncover synergistic effects between TATA and initiator elements, which were masked at mean expression levels. Notably, we provide transcriptome-wide evidence that enhancers control burst frequencies, and demonstrate that cell-type-specific gene expression is primarily shaped by changes in burst frequencies. Together, our data show that burst frequency is primarily encoded in enhancers and burst size in core promoters, and that allelic single-cell RNA sequencing is a powerful model for investigating transcriptional kinetics.Allele-specific single-cell RNA sequencing provides insights into transcription kinetics, with data indicating that core promoter sequences affect burst size, whereas enhancers mainly affect burst frequency., Author(s): Anton J. M. Larsson [sup.1] , Per Johnsson [sup.1] [sup.2] , Michael Hagemann-Jensen [sup.1] , Leonard Hartmanis [sup.1] , Omid R. Faridani [sup.1] [sup.3] , Björn Reinius [sup.1] [sup.2] [...]

Programmed cell death protein-1/ligand-1 (PD-1/PD-L1) blockade is effective in a subset of patients with several tumor types, but predicting patient benefit using approved diagnostics is inexact, as some patients with PD-L1-negative tumors also show clinical benefit.sup.1,2. Moreover, all biopsy-based tests are subject to the errors and limitations of invasive tissue collection.sup.3-11. Preclinical studies of positron-emission tomography (PET) imaging with antibodies to PD-L1 suggested that this imaging method might be an approach to selecting patients.sup.12,13. Such a technique, however, requires substantial clinical development and validation. Here we present the initial results from a first-in-human study to assess the feasibility of imaging with zirconium-89-labeled atezolizumab (anti-PD-L1), including biodistribution, and secondly test its potential to predict response to PD-L1 blockade (ClinicalTrials.gov identifiers NCT02453984 and NCT02478099). We imaged 22 patients across three tumor types before the start of atezolizumab therapy. The PET signal, a function of tracer exposure and target expression, was high in lymphoid tissues and at sites of inflammation. In tumors, uptake was generally high but heterogeneous, varying within and among lesions, patients, and tumor types. Intriguingly, clinical responses in our patients were better correlated with pretreatment PET signal than with immunohistochemistry- or RNA-sequencing-based predictive biomarkers, encouraging further development of molecular PET imaging for assessment of PD-L1 status and clinical response prediction. Initial results from a first-in-human study show that PET imaging with PD-L1 antibodies outperforms immunohistochemistry- or RNA-sequencing-based biomarkers for prediction of clinical response to immunotherapy., Author(s): Frederike Bensch [sup.1] , Elly L. van der Veen [sup.1] , Marjolijn N. Lub-de Hooge [sup.2] [sup.3] , Annelies Jorritsma-Smit [sup.2] , Ronald Boellaard [sup.3] , Iris C. Kok [...]

Epigenomics regulates gene expression and is as important as genomics in precision personal health, as it is heavily influenced by environment and lifestyle. We profiled whole-genome DNA methylation and the corresponding transcriptome of peripheral blood mononuclear cells collected from a human volunteer over a period of 36 months, generating 28 methylome and 57 transcriptome datasets. We found that DNA methylomic changes are associated with infrequent glucose level alteration, whereas the transcriptome underwent dynamic changes during events such as viral infections. Most DNA meta-methylome changes occurred 80-90 days before clinically detectable glucose elevation. Analysis of the deep personal methylome dataset revealed an unprecedented number of allelic differentially methylated regions that remain stable longitudinally and are preferentially associated with allele-specific gene regulation. Our results revealed that changes in different types of 'omics' data associate with different physiological aspects of this individual: DNA methylation with chronic conditions and transcriptome with acute events. Personal 'omics' analysis of a single individual over a period of 3 years reveals that different types of omics data associate with episodes of acute and chronic disease., Author(s): Rui Chen [sup.1] [sup.3] , Lin Xia [sup.2] , Kailing Tu [sup.2] , Meixue Duan [sup.2] , Kimberly Kukurba [sup.1] , Jennifer Li-Pook-Than [sup.1] , Dan Xie [sup.1] [sup.2] [...]

Obesity is a major risk factor for developing nonalcoholic fatty liver disease (NAFLD). NAFLD is the most common form of chronic liver disease and is closely associated with insulin resistance, ultimately leading to cirrhosis and hepatocellular carcinoma. However, knowledge of the intracellular regulators of obesity-linked fatty liver disease remains incomplete. Here we showed that hepatic Rho-kinase 1 (ROCK1) drives obesity-induced steatosis in mice through stimulation of de novo lipogenesis. Mice lacking ROCK1 in the liver were resistant to diet-induced obesity owing to increased energy expenditure and thermogenic gene expression. Constitutive expression of hepatic ROCK1 was sufficient to promote adiposity, insulin resistance, and hepatic lipid accumulation in mice fed a high-fat diet. Correspondingly, liver-specific ROCK1 deletion prevented the development of severe hepatic steatosis and reduced hyperglycemia in obese diabetic (ob/ob) mice. Of pathophysiological significance, hepatic ROCK1 was markedly upregulated in humans with fatty liver disease and correlated with risk factors clustering around NAFLD and insulin resistance. Mechanistically, we found that hepatic ROCK1 suppresses AMPK activity and a ROCK1/AMPK pathway is necessary to mediate cannabinoid-induced lipogenesis in the liver. Furthermore, treatment with metformin, the most widely used antidiabetes drug, reduced hepatic lipid accumulation by inactivating ROCK1, resulting in activation of AMPK downstream signaling. Taken together, our findings establish a ROCK1/AMPK signaling axis that regulates de novo lipogenesis, providing a unique target for treating obesity-related metabolic disorders such as NAFLD., IntroductionObesity has reached epidemic proportions in the United States and worldwide, and is associated with increased risk for type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), atherosclerotic disease, sleep apnea, [...]

The mammalian cochlea contains two types of mechanosensory hair cell that have different and critical functions in hearing. Inner hair cells (IHCs), which have an elaborate presynaptic apparatus, signal to cochlear neurons and communicate sound information to the brain. Outer hair cells (OHCs) mechanically amplify sound-induced vibrations, providing enhanced sensitivity to sound and sharp tuning. Cochlear hair cells are solely generated during development, and hair cell death--most often of OHCs--is the most common cause of deafness. OHCs and IHCs, together with supporting cells, originate in embryos from the prosensory region of the otocyst, but how hair cells differentiate into two different types is unknown.sup.1-3. Here we show that Insm1, which encodes a zinc finger protein that is transiently expressed in nascent OHCs, consolidates their fate by preventing trans-differentiation into IHCs. In the absence of INSM1, many hair cells that are born as OHCs switch fates to become mature IHCs. To identify the genetic mechanisms by which Insm1 operates, we compared the transcriptomes of immature IHCs and OHCs, and of OHCs with and without INSM1. In OHCs that lack INSM1, a set of genes is upregulated, most of which are normally preferentially expressed by IHCs. The homeotic cell transformation of OHCs without INSM1 into IHCs reveals a mechanism by which these neighbouring mechanosensory cells begin to differ: INSM1 represses a core set of early IHC-enriched genes in embryonic OHCs and makes them unresponsive to an IHC-inducing gradient, so that they proceed to mature as OHCs. Without INSM1, some of the OHCs in which these few IHC-enriched transcripts are upregulated trans-differentiate into IHCs, identifying candidate genes for IHC-specific differentiation.Conditional deletion of Insm1 in mice demonstrates that INSM1 is the key switch that causes the maturation of outer hair cells in the cochlea, with its absence resulting in an increase in inner hair cells instead., Author(s): Teerawat Wiwatpanit [sup.1] [sup.2] , Sarah M. Lorenzen [sup.1] [sup.3] , Jorge A. Cantú [sup.1] , Chuan Zhi Foo [sup.1] [sup.2] , Ann K. Hogan [sup.1] [sup.2] , Freddie [...]

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host.sup.1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility.sup.2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding.sup.4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses--Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing--that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.Long-read sequencing allows the assembly of antigen-gene arrays in Trypanosoma brucei and, coupled with deletion experiments, demonstrates that histone variants act as a molecular link between genome architecture, chromatin conformation and antigen variation., Author(s): Laura S. M. Müller [sup.1] [sup.2] [sup.3] , Raúl O. Cosentino [sup.1] [sup.2] [sup.3] , Konrad U. Förstner [sup.4] [sup.5] [sup.6] , Julien Guizetti [sup.3] [sup.14] , Carolin Wedel [...]

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNA.sup.Ala mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin. Mitochondrially targeted zinc-finger nucleases reduce mutational burden and correct biochemical defects in a mouse model of mitochondrial disease., Author(s): Payam A. Gammage [sup.1] , Carlo Viscomi [sup.1] , Marie-Lune Simard [sup.2] , Ana S. H. Costa [sup.3] , Edoardo Gaude [sup.3] , Christopher A. Powell [sup.1] , Lindsey [...]

Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells.sup.1-3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes.sup.3-6 and DNase I hypersensitive sites.sup.7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling.sup.8,9 that may be related to heterogeneity in chromatin accessibility.sup.10-12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.Single-cell micrococcal nuclease sequencing simultaneously measures chromatin accessibility and genome-wide nucleosome positioning in single cells to reveal principles of nucleosome organization., Author(s): Binbin Lai [sup.1] , Weiwu Gao [sup.1] [sup.2] , Kairong Cui [sup.1] , Wanli Xie [sup.1] [sup.3] , Qingsong Tang [sup.1] , Wenfei Jin [sup.4] , Gangqing Hu [sup.1] [...]

The human genome contains thousands of long non-coding RNAs.sup.1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen.sup.2-7. A specific long non-coding RNA--non-coding RNA activated by DNA damage (NORAD)--has recently been shown to be required for maintaining genomic stability.sup.8, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex--which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)--that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression--which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.The long non-coding RNA NORAD interacts with proteins involved in DNA replication and repair, and controls the ability of RBMX to form a ribonucleoprotein complex that helps to maintain genomic stability., Author(s): Mathias Munschauer [sup.1] , Celina T. Nguyen [sup.1] , Klara Sirokman [sup.1] , Christina R. Hartigan [sup.1] , Larson Hogstrom [sup.1] , Jesse M. Engreitz [sup.1] , Jacob C. [...]

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-[kappa]B signalling.sup.1. Recent studies indicate that the bacterial metabolite d-glycero-[beta]-d-manno-heptose 1,7-bisphosphate (HBP) can activate NF-[kappa]B signalling in host cytosol.sup.2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-[beta]-d-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-[kappa]B activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-[kappa]B. A CRISPR-Cas9 screen showed that activation of NF-[kappa]B by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.The bacterial metabolite ADP-heptose activates NF-[kappa]B in host cells via alpha-kinase 1 and the TIFA-TRAF signalling pathway., Author(s): Ping Zhou [sup.1] , Yang She [sup.1] [sup.2] [sup.3] , Na Dong [sup.4] , Peng Li [sup.1] , Huabin He [sup.1] , Alessio Borio [sup.5] , Qingcui Wu [sup.1] [...]

RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput.sup.1. However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity--the time derivative of the gene expression state--can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.RNA velocity, estimated in single cells by comparison of spliced and unspliced mRNA, is a good indicator of transcriptome dynamics and will provide a useful tool for analysis of developmental lineage., Author(s): Gioele La Manno [sup.1] [sup.2] , Ruslan Soldatov [sup.3] , Amit Zeisel [sup.1] [sup.2] , Emelie Braun [sup.1] [sup.2] , Hannah Hochgerner [sup.1] [sup.2] , Viktor Petukhov [sup.3] [sup.4] [...]

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages.sup.1,2. In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death.sup.3, but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML. Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 block AML progression by promoting CARD8-dependent pyroptosis of leukemic myeloid cells., Author(s): Darren C. Johnson [sup.1] , Cornelius Y. Taabazuing [sup.2] , Marian C. Okondo [sup.2] , Ashley J. Chui [sup.1] , Sahana D. Rao [sup.1] , Fiona C. Brown [sup.3] [...]

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease.sup.1,2 and embryonic lethality during mouse development.sup.3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice.sup.4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon. OTULIN, which removes ubiquitin chains deposited by LUBAC, promotes LUBAC activity by preventing its auto-ubiquitination, thereby supporting normal mouse embryo development and preventing pro-inflammatory cell death in adult mice., Author(s): Klaus Heger [sup.1] , Katherine E. Wickliffe [sup.1] , Ada Ndoja [sup.1] , Juan Zhang [sup.2] , Aditya Murthy [sup.3] , Debra L. Dugger [sup.1] , Allie Maltzman [sup.1] [...]

Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function.X-ray and cryo-electron microscopy structures of fungal mitochondrial calcium uniporter proteins reveal a tetrameric architecture and shed light on the function of the channel., Author(s): Chao Fan [sup.1] , Minrui Fan [sup.1] , Benjamin J. Orlando [sup.2] , Nathan M. Fastman [sup.1] [sup.3] , Jinru Zhang [sup.1] , Yan Xu [sup.1] , Melissa G. [...]

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase.sup.1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis.sup.2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production.sup.3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials.sup.4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy.sup.5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen.sup.6,7. This screen yielded FTCD, which encodes an enzyme--formimidoyltransferase cyclodeaminase--that is required for the catabolism of the amino acid histidine.sup.8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.Histidine metabolism influences the sensitivity of cancer cells to methotrexate, with mice bearing leukaemia xenografts showing increased response to the drug upon histidine supplementation., Author(s): Naama Kanarek [sup.1] [sup.2] [sup.3] [sup.4] , Heather R. Keys [sup.1] , Jason R. Cantor [sup.1] [sup.2] [sup.3] [sup.4] , Caroline A. Lewis [sup.1] , Sze Ham Chan [sup.1] [...]

Author(s): P. Trayhurn [sup.1] [sup.2] Author Affiliations: (1) Obesity Biology Unit, University of Liverpool, Liverpool, UK (2) Clore Laboratory, University of Buckingham, Buckingham, UK Many whose research career has been [...]

Gasdermins mediate inflammatory cell death after cleavage by caspases or other, unknown enzymes. The cleaved N-terminal fragments bind to acidic membrane lipids to form pores, but the mechanism of pore formation remains unresolved. Here we present the cryo-electron microscopy structures of the 27-fold and 28-fold single-ring pores formed by the N-terminal fragment of mouse GSDMA3 (GSDMA3-NT) at 3.8 and 4.2 Å resolutions, and of a double-ring pore at 4.6 Å resolution. In the 27-fold pore, a 108-stranded anti-parallel [beta]-barrel is formed by two [beta]-hairpins from each subunit capped by a globular domain. We identify a positively charged helix that interacts with the acidic lipid cardiolipin. GSDMA3-NT undergoes radical conformational changes upon membrane insertion to form long, membrane-spanning [beta]-strands. We also observe an unexpected additional symmetric ring of GSDMA3-NT subunits that does not insert into the membrane in the double-ring pore, which may represent a pre-pore state of GSDMA3-NT. These structures provide a basis that explains the activities of several mutant gasdermins, including defective mutants that are associated with cancer.High-resolution cryo-electron microscopy structures of the membrane-pore-forming domain of the mouse gasdermin GSDMA3 show that it forms pores with 26-, 27- or 28-fold symmetry and indicate that it may also form a parallel, soluble, pre-pore ring structure., Author(s): Jianbin Ruan [sup.1] [sup.2] , Shiyu Xia [sup.1] [sup.2] , Xing Liu [sup.1] [sup.3] , Judy Lieberman [sup.1] [sup.3] , Hao Wu [sup.1] [sup.2] Author Affiliations:(1) Program in Cellular [...]

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1.sup.1. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death.sup.2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype.sup.9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1.sup.-/- (also known as Rbck1.sup.-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3.sup.-/-Casp8.sup.-/-Hoil-1.sup.-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.The HOIL-1 component of the LUBAC ubiquitin ligase complex is required for LUBAC activity, which prevents lethality during embryogenesis by preventing aberrant TNFR1-mediated endothelial cell death and RIPK1-mediated defects in haematopoiesis., Author(s): Nieves Peltzer [sup.1] , Maurice Darding [sup.1] , Antonella Montinaro [sup.1] , Peter Draber [sup.1] [sup.2] , Helena Draberova [sup.1] [sup.2] , Sebastian Kupka [sup.1] , Eva Rieser [sup.1] [...]

Understanding chirality, or handedness, in molecules is important because of the enantioselectivity that is observed in many biochemical reactions.sup.1, and because of the recent development of chiral metamaterials with exceptional light-manipulating capabilities, such as polarization control.sup.2-4, a negative refractive index.sup.5 and chiral sensing.sup.6. Chiral nanostructures have been produced using nanofabrication techniques such as lithography.sup.7 and molecular self-assembly.sup.8-11, but large-scale and simple fabrication methods for three-dimensional chiral structures remain a challenge. In this regard, chirality transfer represents a simpler and more efficient method for controlling chiral morphology.sup.12-18. Although a few studies.sup.18,19 have described the transfer of molecular chirality into micrometre-sized helical ceramic crystals, this technique has yet to be implemented for metal nanoparticles with sizes of hundreds of nanometres. Here we develop a strategy for synthesizing chiral gold nanoparticles that involves using amino acids and peptides to control the optical activity, handedness and chiral plasmonic resonance of the nanoparticles. The key requirement for achieving such chiral structures is the formation of high-Miller-index surfaces ({hkl}, h [not equal to] k [not equal to] l [not equal to] 0) that are intrinsically chiral, owing to the presence of 'kink' sites.sup.20-22 in the nanoparticles during growth. The presence of chiral components at the inorganic surface of the nanoparticles and in the amino acids and peptides results in enantioselective interactions at the interface between these elements; these interactions lead to asymmetric evolution of the nanoparticles and the formation of helicoid morphologies that consist of highly twisted chiral elements. The gold nanoparticles that we grow display strong chiral plasmonic optical activity (a dis-symmetry factor of 0.2), even when dispersed randomly in solution; this observation is supported by theoretical calculations and direct visualizations of macroscopic colour transformations. We anticipate that our strategy will aid in the rational design and fabrication of three-dimensional chiral nanostructures for use in plasmonic metamaterial applications. Chirality can be 'encoded' into gold nanoparticles by introducing chiral amino acids or peptides during the growth process, leading to the formation of helicoid morphologies., Author(s): Hye-Eun Lee [sup.1] , Hyo-Yong Ahn [sup.1] , Jungho Mun [sup.2] , Yoon Young Lee [sup.1] , Minkyung Kim [sup.3] , Nam Heon Cho [sup.1] , Kiseok Chang [sup.4] [...]

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines.sup.1. Biochemical studies.sup.2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2--a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)--in complex with nucleosomes.sup.5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding.sup.8, histone exchange.sup.9 and nucleosome spacing.sup.10-12 remain poorly understood. Although some remodellers work as monomers.sup.13, others work as highly cooperative dimers.sup.11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain--unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains. Cryo-electron microscopy structure of the human INO80 chromatin remodeller in complex with a bound nucleosome reveals that its motor domains are located at the DNA wrap around the histone core., Author(s): Rafael Ayala [sup.1] , Oliver Willhoft [sup.1] , Ricardo J. Aramayo [sup.1] , Martin Wilkinson [sup.1] , Elizabeth A. McCormack [sup.1] , Lorraine Ocloo [sup.1] , Dale B. Wigley [...]

A desire for a simpler, cheaper way to do common laboratory tests for medical diagnoses and to avoid 'washing the dishes' led UConn researchers to develop a new technology that [...]

Antitumor immunity can be enhanced by blocking NKG2A, an inhibitory receptor expressed on natural killer and T cells., Author(s): Matthew D. Hellmann [sup.1] [sup.2] , Alexandra Snyder [sup.3] Author Affiliations: (1) Memorial Sloan Kettering Cancer Center, New York, USA (2) Weill Cornell Medical College, New York, USA (3) [...]

Background: Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects. Objectives: Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects. Methods: Female mice were exposed to GEN on postnatal days (PND)1-5 and uterine tissues collected on PND5, PND22-26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to 17[beta]-estradiol (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators. Results: GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including Foxa2, Wnt4, and Sox17. These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of Foxa2, Wnt4, and Sox17, as well as genes important for neonatal uterine differentiation (Wnt7a, Hoxa10, and Msx2), were severely disrupted on PND5 in GEN-exposed mice. Discussion: Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants., Introduction Developmental exposure to estrogenic chemicals is associated with abnormalities in the female reproductive tract that lead to infertility and cancer in women and in mouse models (Reed and Fenton [...]