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Thermoplastic parts manufactured via fused filament fabrication (FFF) have limited strength and toughness compared to other types of polymer additive and subtractive manufacturing. Low strength results from poor interlayer adhesion, making FFF parts not suitable for most engineering applications. Post processing solutions, such as annealing, enable healing of these interlayers, thus approaching injection molded parts. Prior work demonstrated a core-shell polycarbonate (PC)-acrylonitrile butadiene styrene (ABS) structured dual material filament to provide thermo-structural stability during annealing of the ABS component; however, annealing was limited to relatively low temperatures (135 °C) and required long annealing times (72 h). In the current work, a PC copolymer with a higher glass transition temperature (173 °C) than conventional PC is processed along with an extrusion-grade ABS into a PC-ABS core-shell filament. This improved dual material filament was printed, annealed, and evaluated via Izod impact testing, ultimately yielding 83% of bulk annealed ABS z-direction strength at an accelerated annealing time (8 h) and higher annealing temperature (155-175 °C). A demonstration part is printed with the dual material filament and annealed at 155 °C for 8 h, resulting in excellent dimensional accuracy, and a ductile failure at 73% higher ultimate load compared to the brittle failure of an as-printed part. This work highlights that material selection and design of a bicomponent filament geometry can lead to parts printed with FFF, with increased strength compared to other post-processing techniques at reduced processing times.
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BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.
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Colite , Doença de Crohn , Animais , Doença de Crohn/genética , Doença de Crohn/patologia , Constrição Patológica , Intestinos/patologia , Colite/patologia , Fibroblastos/patologiaRESUMO
Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-ß1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-ß1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-ß1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-ß1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.
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Fator Estimulador de Colônias de Granulócitos e Macrófagos , Fibrose Pulmonar , Humanos , Camundongos , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Interleucina-17/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Cicatriz , Macrófagos/patologia , Inflamação/patologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Glicoproteínas de Membrana , Receptores ImunológicosRESUMO
Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.
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[This corrects the article DOI: 10.1007/s12195-021-000689-6.].
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INTRODUCTION: Mechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. METHODS: We show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains. RESULTS: As an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). CONCLUSIONS: This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00689-6.
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Productive transmission of malaria parasites hinges upon the execution of key transcriptional and posttranscriptional regulatory events. While much is now known about how specific transcription factors activate or repress sexual commitment programs, far less is known about the production of a preferred mRNA homeostasis following commitment and through the host-to-vector transmission event. Here, we show that in Plasmodium parasites, the NOT1 scaffold protein of the CAF1/CCR4/Not complex is duplicated, and one paralogue is dedicated for essential transmission functions. Moreover, this NOT1-G paralogue is central to the sex-specific functions previously associated with its interacting partners, as deletion of not1-g in Plasmodium yoelii leads to a comparable or complete arrest phenotype for both male and female parasites. We show that, consistent with its role in other eukaryotes, PyNOT1-G localizes to cytosolic puncta throughout much of the Plasmodium life cycle. PyNOT1-G is essential to both the complete maturation of male gametes and to the continued development of the fertilized zygote originating from female parasites. Comparative transcriptomics of wild-type and pynot1-g- parasites shows that loss of PyNOT1-G leads to transcript dysregulation preceding and during gametocytogenesis and shows that PyNOT1-G acts to preserve mRNAs that are critical to sexual and early mosquito stage development. Finally, we demonstrate that the tristetraprolin (TTP)-binding domain, which acts as the typical organization platform for RNA decay (TTP) and RNA preservation (ELAV/HuR) factors is dispensable for PyNOT1-G's essential blood stage functions but impacts host-to-vector transmission. Together, we conclude that a NOT1-G paralogue in Plasmodium fulfills the complex transmission requirements of both male and female parasites.
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Estágios do Ciclo de Vida , Parasitos/crescimento & desenvolvimento , Parasitos/metabolismo , Plasmodium/crescimento & desenvolvimento , Plasmodium/metabolismo , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Animais , Citosol/metabolismo , Feminino , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/fisiologia , Masculino , Camundongos , Modelos Biológicos , Domínios Proteicos , Mapas de Interação de Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Maturidade Sexual/fisiologia , Transcriptoma/genética , Zigoto/crescimento & desenvolvimentoRESUMO
In recent years, a number of groups have been investigating the use of "empty" liposomes with no drug loaded as scavengers both for exogenous intoxicants and endogenous toxic molecules. Preclinical trials have demonstrated that repurposing liposomes to sequester such compounds may prove clinically useful. The use of such "empty" liposomes in the dialysate during dialysis avoids recognition by complement surveillance, allowing high doses of liposomes to be used. The "reach" of dialysis may also be increased to molecules that are not traditionally dialysable. We aim to review the current literature in this area with the aims of increasing awareness and informing further research. A structured literature search identified thirteen papers which met the inclusion criteria. Augmenting the extraction of ammonia in hepatic failure with pH-gradient liposomes with acidic centres in peritoneal dialysis is the most studied area, with work progressing toward phase one trials. Liposomes used to augment the removal of exogenous intoxicants and protein-bound uraemic and hepatic toxins that accumulate in these organ failures and liposome-supported enzymatic dialysis have also been studied. It is conceivable that liposomes will be repurposed from the role of pharmaceutical vectors to gain further indications as clinically useful nanomedical antidotes/treatments within the next decade.
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BACKGROUND: Well-defined promoters are essential elements for genetic studies in all organisms, and enable controlled expression of endogenous genes, transgene expression, and gene editing. Despite this, there is a paucity of defined promoters for the rodent-infectious malaria parasites. This is especially true for Plasmodium yoelii, which is often used to study the mosquito and liver stages of malarial infection, as well as host immune responses to infection. METHODS: Here six promoters were selected from across the parasite's life cycle (clag-a, dynein heavy chain delta, lap4, trap, uis4, lisp2) that have been invoked in the literature as controlling their genes in a stage-specific manner. A minimal promoter length for the constitutive pybip promoter that confers strong expression levels was also determined, which is useful for expression of reporters and gene editing enzymes. RESULTS: Instead, it was observed that these promoters confer stage-enriched gene control, as some parasites also effectively use these promoters in other stages. Thus, when used alone, these promoters could complicate the interpretation of results obtained from promoter swaps, stage-targeted recombination, or gene editing experiments. CONCLUSIONS: Together these data indicate that achieving stage-specific effects, such as gene editing, is likely best done using a two-component system with independent promoter activities overlapping only in the intended life cycle stage.
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Genes de Protozoários , Malária/fisiopatologia , Plasmodium yoelii/genética , Regiões Promotoras Genéticas , Animais , Feminino , CamundongosRESUMO
Plasmodium sporozoites are transmitted from infected mosquitoes to mammals, and must navigate the host skin and vasculature to infect the liver. This journey requires distinct proteomes. Here, we report the dynamic transcriptomes and proteomes of both oocyst sporozoites and salivary gland sporozoites in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. The data robustly define mRNAs and proteins that are upregulated in oocyst sporozoites (UOS) or upregulated in infectious sporozoites (UIS) within the salivary glands, including many that are essential for sporozoite functions in the vector and host. Moreover, we find that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression. Together with gene-specific validation experiments, these data indicate that two waves of translational repression are implemented and relieved at different times during sporozoite maturation, migration and infection, thus promoting their successful development and vector-to-host transition.
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Regulação da Expressão Gênica no Desenvolvimento/genética , Oocistos/genética , Plasmodium falciparum/genética , Plasmodium yoelii/genética , Proteoma/metabolismo , RNA Mensageiro/metabolismo , Esporozoítos/genética , Transcriptoma/genética , Animais , Anopheles/parasitologia , Cromatografia Líquida , Repressão Epigenética/genética , Perfilação da Expressão Gênica , Humanos , Malária , Malária Falciparum , Mosquitos Vetores/parasitologia , Oocistos/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium yoelii/metabolismo , Proteômica , Roedores , Glândulas Salivares/parasitologia , Esporozoítos/metabolismo , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites may rely more on the regulation of transcript stability and turnover to provide sufficient gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized Plasmodium yoelii (Py) CCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that deletion of the pyccr4-1 gene does not result in gross changes to its UTR or poly(A) tail length. We conclude that the two putative deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.
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Plasmodium falciparum/genética , Receptores CCR4/metabolismo , Animais , Culicidae/metabolismo , Exorribonucleases , Gametogênese/fisiologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio , Masculino , Camundongos , Mosquitos Vetores , Plasmodium/genética , Plasmodium falciparum/metabolismo , Proteínas , RNA Mensageiro/genética , Proteínas Repressoras , Ribonucleases , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Fibroproliferative diseases affect a significant proportion of the world's population. Despite this, core mechanisms driving organ fibrosis of diverse etiologies remain ill defined. Recent studies suggest that integrin-alpha V serves as a master driver of fibrosis in multiple organs. Although diverse mechanisms contribute to the progression of fibrosis, TGF-ß and IL-13 have emerged as central mediators of fibrosis during type 1/type 17, and type 2 polarized inflammatory responses, respectively. To investigate if integrin-alpha V interactions or signaling is critical to the development of type 2 fibrosis, we analyzed fibroblast-specific integrin-alpha V knockout mice in three type 2-driven inflammatory disease models. While we confirmed a role for integrin-alpha V in type 17-associated fibrosis, integrin-alpha V was not critical to the development of type 2-driven fibrosis. Additionally, our studies support a novel mechanism through which fibroblasts, via integrin-alpha V expression, are capable of regulating immune polarization. We show that when integrin-alpha V is deleted on fibroblasts, initiation of type 17 inflammation is inhibited leading to a deregulation of type 2 inflammation. This mechanism is most evident in a model of severe asthma, which is characterized by a mixed type 2/type 17 inflammatory response. Together, these findings suggest dual targeting of integrin-alpha V and type 2 pathways may be needed to ameliorate fibrosis and prevent rebound of opposing pro-fibrotic and inflammatory mechanisms. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Fibroblastos/metabolismo , Inflamação/metabolismo , Integrina alfa5/fisiologia , Animais , Asma/metabolismo , Asma/prevenção & controle , Modelos Animais de Doenças , Feminino , Fibrose , Deleção de Genes , Inflamação/patologia , Integrina alfa5/genética , Interleucina-13/antagonistas & inibidores , Interleucina-13/imunologia , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Masculino , Camundongos Knockout , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Fibrose Pulmonar/prevenção & controleRESUMO
In this study, we characterized the Puf family gene member Puf3 in the malaria parasites Plasmodium falciparum and Plasmodium yoelii Secondary structure prediction suggested that the RNA-binding domains of the Puf3 proteins consisted of 11 pumilio repeats that were similar to those in the human Puf-A (also known as PUM3) and Saccharomyces cerevisiae Puf6 proteins, which are involved in ribosome biogenesis. Neither P. falciparum (Pf)Puf3 nor P. yoelii (Py)Puf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle. Cellular fractionation of PfPuf3 in the asexual stages revealed preferential partitioning to the nuclear fraction, consistent with nuclear localization of PfPuf3::GFP and PyPuf3::GFP as detected by immunofluorescence. Furthermore, PfPuf3 colocalized with the nucleolar marker PfNop1, demonstrating that PfPuf3 is a nucleolar protein in the asexual stages. We found, however, that PyPuf3 changed its localization from being nucleolar to being present in cytosolic puncta in the mosquito and liver stages, which may reflect alternative functions in these stages. Affinity purification of molecules that associated with a PTP-tagged variant of PfPuf3 revealed 31 proteins associated with the 60S ribosome, and an enrichment of 28S rRNA and internal transcribed spacer 2 sequences. Taken together, these results suggest an essential function for PfPuf3 in ribosomal biogenesis.
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Plasmodium falciparum/metabolismo , Plasmodium yoelii/metabolismo , Proteínas de Protozoários/química , Ribossomos/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Citosol/metabolismo , Estágios do Ciclo de Vida , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium yoelii/química , Plasmodium yoelii/genética , Plasmodium yoelii/crescimento & desenvolvimento , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Malaria is a devastating illness that causes approximately 500,000 deaths annually. The malaria-causing parasite (Plasmodium genus) uses the process of translational repression to regulate its growth, development, and transmission. As poly(A)-binding proteins (PABP) have been identified as critical components of RNA metabolism and translational repression in model eukaryotes and in Plasmodium, we have identified and investigated two PABPs in Plasmodium yoelii, PyPABP1 and PyPABP2. In contrast to most single-celled eukaryotes, Plasmodium closely resembles metazoans and encodes both a nuclear PABP and a cytosolic PABP; here, we provide multiple lines of evidence in support of this observation. The conserved domain architectures of PyPABP1 and PyPABP2 resemble those of yeast and metazoans, while multiple independent binding assays demonstrated their ability to bind very strongly and specifically to poly(A) sequences. Interestingly, we also observed that purified PyPABP1 forms homopolymeric chains despite exhaustive RNase treatment in vitro. Finally, we show by indirect immunofluorescence assays (IFAs) that PyPABP1 and PyPABP2 are cytoplasm- and nucleus-associated PABPs during the blood stages of the life cycle. Surprisingly, however, PyPABP1 was instead observed to also be localized on the surface of transmitted salivary gland sporozoites and to be deposited in trails when parasites glide on a substrate. This is the third RNA-binding protein verified to be found on the sporozoite surface, and the data may point to an unappreciated RNA-centered interface between the host and parasite. IMPORTANCE Malaria remains one of the great global health problems. The parasite that causes malaria (Plasmodium genus) relies upon exquisite control of its transmission between vertebrate hosts and mosquitoes. One crucial way that it does so is by proactively producing mRNAs needed to establish the new infection but by silencing and storing them until they are needed. One key protein in this process of translational repression in model eukaryotes is poly(A)-binding protein (PABP). Here we have shown that Plasmodium yoelii utilizes both a nuclear PABP and a cytosolic PABP, both of which bind specifically to polyadenylated RNA sequences. Moreover, we find that the cytosolic PABP forms chains in vitro, consistent with its appreciated role in coating the poly(A) tails of mRNA. Finally, we have also verified that, surprisingly, the cytosolic PABP is found on the surface of Plasmodium sporozoites. Taking the data together, we propose that Plasmodium utilizes a more metazoan-like strategy for RNA metabolism using specialized PABPs.
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Transmission of the malaria parasite occurs in an unpredictable moment, when a mosquito takes a blood meal. Plasmodium has therefore evolved strategies to prepare for transmission, including translationally repressing and protecting mRNAs needed to establish the infection. However, mechanisms underlying these critical controls are not well understood, including whether Plasmodium changes its translationally repressive complexes and mRNA targets in different stages. Efforts to understand this have been stymied by severe technical limitations due to substantial mosquito contamination of samples. Here using P. yoelii, for the first time we provide a proteomic comparison of a protein complex across asexual blood, sexual and sporozoite stages, along with a transcriptomic comparison of the mRNAs that are affected in these stages. We find that the Apicomplexan-specific ALBA4 RNA-binding protein acts to regulate development of the parasite's transmission stages, and that ALBA4 associates with both stage-specific and stage-independent partners to produce opposing mRNA fates. These efforts expand our understanding and ability to interrogate both sexual and sporozoite transmission stages and the molecular preparations they evolved to perpetuate their infectious cycle.
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Plasmodium yoelii/fisiologia , RNA Mensageiro/biossíntese , Animais , Anopheles/parasitologia , Repressão Enzimática , Malária/parasitologia , Parasitos , Doenças Parasitárias/genética , Plasmodium yoelii/genética , Plasmodium yoelii/crescimento & desenvolvimento , Proteômica , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Esporozoítos/metabolismo , TranscriptomaRESUMO
Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.
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Caderinas/metabolismo , Células Epiteliais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Adesão Celular/fisiologia , Divisão Celular , Forma Celular , Cães , Células Epiteliais/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células Madin Darby de Rim Canino , Mecanotransdução Celular , Fuso Acromático/metabolismo , Estresse Mecânico , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countries and is the second leading indication for liver transplantation due to the extensive fibrosis it causes. NAFLD progression is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity; however, the specific immunological mechanisms regulating the progression of NAFLD-associated fibrosis in the liver are unclear. To investigate the immunopathogenesis of NAFLD more completely, we investigated adipose dysfunction, nonalcoholic steatohepatitis (NASH), and fibrosis in mice that develop polarized type 1 or type 2 immune responses. Unexpectedly, obese interleukin-10 (IL-10)/IL-4-deficient mice (type 1-polarized) were highly resistant to NASH. This protection was associated with an increased hepatic interferon-γ (IFN-γ) signature. Conversely, IFN-γ-deficient mice progressed rapidly to NASH with evidence of fibrosis dependent on transforming growth factor-ß (TGF-ß) and IL-13 signaling. Unlike increasing type 1 inflammation and the marked loss of eosinophils seen in expanding adipose tissue, progression of NASH was associated with increasing eosinophilic type 2 liver inflammation in mice and human patient biopsies. Finally, simultaneous inhibition of TGF-ß and IL-13 signaling attenuated the fibrotic machinery more completely than TGF-ß alone in NAFLD-associated fibrosis. Thus, although type 2 immunity maintains healthy metabolic signaling in adipose tissues, it exacerbates the progression of NAFLD collaboratively with TGF-ß in the liver.
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Progressão da Doença , Imunidade , Doenças Metabólicas/imunologia , Doenças Metabólicas/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Fator de Crescimento Transformador beta/metabolismo , Tecido Adiposo/patologia , Animais , Dieta Hiperlipídica , Eosinófilos/patologia , Humanos , Inflamação/patologia , Interferon gama/deficiência , Interferon gama/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/patologiaRESUMO
The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.