Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages.

Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to postfusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we harness the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. We show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability and shelf-life. Furthermore, it has 11X greater potency as compared with the DS-Cav1 stabilized prefusion F protein in immunogenicity studies and overcomes immunosenescence in mice with simply a high-dose formulation on alum.

Prolactin receptor signaling induces acquisition of chemoresistance and reduces clonogenicity in acute myeloid leukemia.

BACKGROUND: Development of precision medicine requires the identification of easily detectable and druggable biomarkers. Despite recent targeted drug approvals, prognosis of acute myeloid leukemia (AML) patients needs to be greatly improved, as relapse and refractory disease are still difficult to manage. Thus, new therapeutic approaches are needed. Based on in silico-generated preliminary data and the literature, the role of the prolactin (PRL)-mediated signaling was interrogated in AML. METHODS: Protein expression and cell viability were determined by flow cytometry. Repopulation capacity was studied in murine xenotransplantation assays. Gene expression was measured by qPCR and luciferase-reporters. SA-ß-Gal staining was used as a senescence marker. RESULTS: The prolactin receptor (PRLR) was upregulated in AML cells, as compared to their healthy counterpart. The genetic and molecular inhibition of this receptor reduced the colony-forming potential. Disruption of the PRLR signaling, either using a mutant PRL or a dominant-negative isoform of PRLR, reduced the leukemia burden in vivo, in xenotransplantation assays. The expression levels of PRLR directly correlated with resistance to cytarabine. Indeed, acquired cytarabine resistance was accompanied with the induction of PRLR surface expression. The signaling associated to PRLR in AML was mainly mediated by Stat5, in contrast to the residual function of Stat3. In concordance, Stat5 mRNA was significantly overexpressed at mRNA levels in relapse AML samples. A senescence-like phenotype, measured by SA-ß-gal staining, was induced upon enforced expression of PRLR in AML cells, partially dependent on ATR. Similar to the previously described chemoresistance-induced senescence in AML, no cell cycle arrest was observed. Additionally, the therapeutic potential of PRLR in AML was genetically validated. CONCLUSIONS: These results support the role of PRLR as a therapeutic target for AML and the further development of drug discovery programs searching for specific PRLR inhibitors.

A Novel Family of Lysosomotropic Tetracyclic Compounds for Treating Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological cancer characterized by poor prognosis and frequent relapses. Aside from specific mutation-related changes, in AML, the overall function of lysosomes and mitochondria is drastically altered to fulfill the elevated biomass and bioenergetic demands. On the basis of previous results, in silico drug discovery screening was used to identify a new family of lysosome-/mitochondria-targeting compounds. These novel tetracyclic hits, with a cationic amphiphilic structure, specifically eradicate leukemic cells by inducing both mitochondrial damage and apoptosis, and simultaneous lysosomal membrane leakiness. Lysosomal leakiness does not only elicit canonical lysosome-dependent cell death, but also activates the terminal differentiation of AML cells through the Ca2+-TFEB-MYC signaling axis. In addition to being an effective monotherapy, its combination with the chemotherapeutic arsenic trioxide (ATO) used in other types of leukemia is highly synergistic in AML cells, widening the therapeutic window of the treatment. Moreover, the compounds are effective in a wide panel of cancer cell lines and possess adequate pharmacological properties rendering them promising drug candidates for the treatment of AML and other neoplasias.

Using the Synergy between HPLC-MS and MALDI-MS Imaging to Explore the Lipidomics of Clear Cell Renal Cell Carcinoma.

Lipid imaging mass spectrometry (LIMS) has been tested in several pathological contexts, demonstrating its ability to segregate and isolate lipid signatures in complex tissues, thanks to the technique's spatial resolution. However, it cannot yet compete with the superior identification power of high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), and therefore, very often, the latter is used to refine the assignment of the species detected by LIMS. Also, it is not clear if the differences in sensitivity and spatial resolution between the two techniques lead to a similar panel of biomarkers for a given disease. Here, we explore the capabilities of LIMS and HPLC-MS to produce a panel of lipid biomarkers to screen nephrectomy samples from 40 clear cell renal cell carcinoma patients. The same set of samples was explored by both techniques, and despite the important differences between them in terms of the number of detected and identified species (148 by LIMS and 344 by HPLC-MS in negative-ion mode) and the presence/absence of image capabilities, similar conclusions were reached: using the lipid fingerprint, it is possible to set up classifiers that correctly identify the samples as either healthy or tumor samples. The spatial resolution of LIMS enables extraction of additional information, such as the existence of necrotic areas or the existence of different tumor cell populations, but such information does not seem determinant for the correct classification of the samples, or it may be somehow compensated by the higher analytical power of HPLC-MS. Similar conclusions were reached with two very different techniques, validating their use for the discovery of lipid biomarkers.

Lysosome-mediated chemoresistance in acute myeloid leukemia.

Despite the outstanding advances in understanding the biology underlying the pathophysiology of acute myeloid leukemia (AML) and the promising preclinical data published lastly, AML treatment still relies on a classic chemotherapy regimen largely unchanged for the past five decades. Recently, new drugs have been approved for AML, but the real clinical benefit is still under evaluation. Nevertheless, primary refractory and relapse AML continue to represent the main clinical challenge, as the majority of AML patients will succumb to the disease despite achieving a complete remission during the induction phase. As such, treatments for chemoresistant AML represent an unmet need in this disease. Although great efforts have been made to decipher the biological basis for leukemogenesis, the mechanism by which AML cells become resistant to chemotherapy is largely unknown. The identification of the signaling pathways involved in resistance may lead to new combinatory therapies or new therapeutic approaches suitable for this subset of patients. Several mechanisms of chemoresistance have been identified, including drug transporters, key secondary messengers, and metabolic regulators. However, no therapeutic approach targeting chemoresistance has succeeded in clinical trials, especially due to broad secondary effects in healthy cells. Recent research has highlighted the importance of lysosomes in this phenomenon. Lysosomes' key role in resistance to chemotherapy includes the potential to sequester drugs, central metabolic signaling role, and gene expression regulation. These results provide further evidence to support the development of new therapeutic approaches that target lysosomes in AML.

LRP1 loss in airway epithelium exacerbates smoke-induced oxidative damage and airway remodeling.

The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice. In vitro, LRP1 knockdown decreased cell migration and increased transforming growth factor ß activation. Tamoxifen-inducible airway-specific LRP1 knockout mice (club Lrp1-/-) induced after complete lung development had increased inflammation in the bronchoalveolar space and lung parenchyma at baseline. After 6 months of smoke exposure, club Lrp1-/- mice showed a combined restrictive and obstructive phenotype, with lower compliance, inspiratory capacity, and forced expiratory volume0.05/forced vital capacity than WT smoke-exposed mice. This was associated with increased values of Ashcroft fibrotic index. Proteomic analysis of room air exposed-club Lrp1-/- mice showed significantly decreased levels of proteins involved in cytoskeleton signaling and xenobiotic detoxification as well as decreased levels of glutathione. The proteome fingerprint created by smoke eclipsed many of the original differences, but club Lrp1-/- mice continued to have decreased lung glutathione levels and increased protein oxidative damage and airway cell proliferation. Therefore, LRP1 deficiency leads to greater lung inflammation and damage and exacerbates smoke-induced lung disease.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Osimertinib-resistant NSCLC cells activate ERBB2 and YAP/TAZ and are killed by neratinib.

We performed additional mechanistic analyses to redefine neratinib biology and determined the mechanisms by which the multi-kinase inhibitor neratinib interacted with the thymidylate synthase inhibitor pemetrexed to kill NSCLC cells expressing either mutant KRAS (G12S; Q61H; G12A; G12C) or mutant NRAS (Q61K) or mutant ERBB1 (L858R; L858R T790M; exon 19 deletion). Neratinib rapidly reduced KRASG12V and RAC1G12V nanoclustering which was followed by KRASG12V, but not RAC1G12V, being extensively mislocalized away from the plasma membrane. This correlated with reduced levels of, and reorganized membrane localization of phosphatidylserine and cholesterol. Reduced nanoclustering was not associated with inactivation of ERBB1, Merlin or Ezrin. The drug combination killed cells expressing mutant KRAS, NRAS or mutant ERBB1 proteins. Afatinib or osimertinib resistant cells were killed with a similar efficacy to non-resistant cells. Compared to osimertinib-resistant cells, sensitive cells had less ERBB2 Y1248 phosphorylation. In osimertinib resistant H1975 cells, the drug combination was less capable of inactivating AKT, mTOR, STAT3, STAT5, ERK1/2 whereas it gained the ability to inactivate ERBB3. In resistant H1650 cells, the drug combination was less capable of inactivating JAK2 and STAT5. Sensitive cells exhibited elevated basal phosphorylation of YAP and TAZ. In resistant cells, portions of YAP and TAZ were localized in the nucleus. [Neratinib + pemetrexed] increased phosphorylation of YAP and TAZ, caused their nuclear exit, and enhanced ERBB2 degradation. Thus, neratinib targets an unidentified protein whose functional inhibition directly results in RAS inactivation and tumor cell killing. Our data prove that, albeit indirectly, oncogenic RAS proteins are druggable by neratinib.

IRGM1 links mitochondrial quality control to autoimmunity.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Autophagy-related protein PIK3C3/VPS34 controls T cell metabolism and function.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is a key early player in macroautophagy/autophagy. In this study, we assessed the contribution of PIK3C3 to T cell metabolism and function. We found that Pik3c3-deficient T cells exhibited impaired cellular metabolism, and Pik3c3-deficient CD4+ T cells failed to differentiate into T helper 1 cells. These alterations were associated with reduced levels of active mitochondria upon T cell activation. In addition, conditional Pik3c3-deficient animals failed to mount autoreactive T cell responses and were resistant to experimental autoimmune encephalomyelitis (EAE). Interestingly, the deletion of Pik3c3 had little effect on the capacity of animals to clear tumor metastases. Collectively, our studies have revealed a critical role of PIK3C3 in T cell metabolism and the pathogenicity of these cells during EAE. Our findings also have important implications for the development of immunotherapies to treat multiple sclerosis and other inflammatory diseases by targeting PIK3C3.Abbreviations: CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; ECAR: extracellular acidification rate; iNKT: invariant natural killer T; LAP: LC3-associated phagocytosis; LLC: Lewis lung carcinoma; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDSC: myeloid-derived suppressor cell; MOG: myelin oligodendrocyte glycoprotein; NK: natural killer; OCR: oxygen consumption rate; PI: propidium iodide; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RNA-seq: RNA-sequencing; TCR: T cell receptor; TMRE: tetramethylrhodamine ethyl ester perchlorate.

Estrogen receptor α phosphorylated at Ser216 confers inflammatory function to mouse microglia.

BACKGROUND: Estrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves the phosphorylation motif at Ser216 within the DNA binding domain. Previously, Ser216 was found to be phosphorylated in neutrophils infiltrating into the mouse uterus and to enable ERα to regulate migration. Given the implication of this phosphorylation in immune regulation, ERα was examined in mouse microglia to determine if Ser216 is phosphorylated and regulates microglia's inflammation. It was found that Ser216 was constitutively phosphorylated in microglia and demonstrated that in the absence of phosphorylated ERα in ERα KI brains microglia inflamed, confirming that phosphorylation confers ERα with anti-inflammatory capability. ERα KI mice were obese and weakened motor ability. METHODS: Mixed glia cells were prepared from brains of 2-days-old neonates and cultured to mature and isolate microglia. An antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used to detect phosphorylated ERα in double immunofluorescence staining with ERα antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα S216A mutation (ERα KI) was generated to examine inflammation-regulating functions of phosphorylated ERα in microglia. RT-PCR, antibody array, ELISA and FACS assays were employed to measure expressions of pro- or anti-inflammatory cytokines at their mRNA and protein levels. Rotarod tests were performed to examine motor connection ability. RESULTS: Double immune staining of mixed glia cells showed that ERα is phosphorylated at Ser216 in microglia, but not astrocytes. Immunohistochemistry with an anti-Iba-1 antibody showed that microglia cells were swollen and shortened branches in the substantial nigra (SN) of ERα KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERα WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERα KI brains as well as cultured microglia, whereas anti-inflammatory cytokines were down-regulated. FACS analysis showed that the number of IL-6 producing and apoptotic microglia increased in those prepared from ERα KI brains. Times of ERα KI mice on rod were shortened in Rotarod tests. CONCLUSIONS: Blocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ERα exerts anti-inflammatory functions. ERα KI mice enable us to further investigate the mechanism by which phosphorylated ERα regulates brain immunity and inflammation and brain diseases. Video abstract.

Detection of LC3-Associated Phagocytosis (LAP).

Phagocytes, notably macrophages, are critical sentinels of their environment, patrolling for and eradicating unwanted components. The ability of cells to process extracellular cargo in an appropriate manner is important for both clearance of the cargo and eventual return to homeostasis. Although the evolutionarily conserved pathway of autophagy involves the degradation and recycling of unnecessary or dysfunctional cellular components during starvation, we now appreciate that the reach of autophagy extends beyond nutrient deprivation, notably including cellular quality control (e.g., mitophagy) and host defense against internalized pathogens (i.e., xenophagy). Despite being seemingly disparate, autophagic functions are unified as conserved mechanisms for containment and immunosuppression, suggesting an original immune function for autophagy. A recently described pathway called LC3-associated phagocytosis (LAP) marries the ancient concepts of phagocytosis and autophagy, revealing new ways in which the autophagy machinery, in a molecularly distinct pathway, contributes to the inflammatory response. In this article, protocols to detect LAP by electron microscopy, immunofluorescence, flow cytometry, and phagosome purification are described, allowing the user to detect multiple characteristics of LAP in both qualitative and quantitative manners. Published 2020. U.S. Government. Basic Protocol 1: Detection of LAP by electron microscopy Basic Protocol 2: Detection of LAP by confocal microscopy of LC3-GFP-expressing cells Alternate Protocol 1: Detection of LAP by confocal microscopy using immunofluorescence Basic Protocol 3: Detection of LAP using flow cytometry of LC3-GFP-expressing cells Alternate Protocol 2: Detection of LAP using antibody staining and flow cytometry Basic Protocol 4: Detection of LAP by western blot of purified LAPosomes.

The clearance of dead cells by efferocytosis.

Multiple modes of cell death have been identified, each with a unique function and each induced in a setting-dependent manner. As billions of cells die during mammalian embryogenesis and daily in adult organisms, clearing dead cells and associated cellular debris is important in physiology. In this Review, we present an overview of the phagocytosis of dead and dying cells, a process known as efferocytosis. Efferocytosis is performed by macrophages and to a lesser extent by other 'professional' phagocytes (such as monocytes and dendritic cells) and 'non-professional' phagocytes, such as epithelial cells. Recent discoveries have shed light on this process and how it functions to maintain tissue homeostasis, tissue repair and organismal health. Here, we outline the mechanisms of efferocytosis, from the recognition of dying cells through to phagocytic engulfment and homeostatic resolution, and highlight the pathophysiological consequences that can arise when this process is abrogated.

Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling.

The irreversible ERBB1/2/4 inhibitor neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles liminal to the plasma membrane; this effect is enhanced by HDAC inhibitors and is now a Phase I trial (NCT03919292). The combination of neratinib and HDAC inhibitors killed pancreatic cancer and lymphoma T cells. Neratinib plus HDAC inhibitor exposure was as efficacious as (paclitaxel+gemcitabine) at killing pancreatic cancer cells. Neratinib reduced the phosphorylation of PAK1, Merlin, LATS1/2, AKT, mTOR, p70 S6K, and ERK1/2 which required expression of Rubicon, Beclin1, and Merlin. Neratinib altered pancreatic tumor cell morphology which was associated with MST4 degradation reduced Ezrin phosphorylation and enhanced phosphorylation of MAP4K4 and LATS1/2. Knockdown of the MAP4K4 activator and sensor of membrane rigidity RAP2A reduced basal LATS1/2 and YAP phosphorylation but did not prevent neratinib from stimulating LATS1/2 or YAP phosphorylation. Beclin1 knockdown prevented MST4 degradation, Ezrin dephosphorylation and neratinib-induced alterations in tumor cell morphology. Our findings demonstrate that neratinib enhances LATS1/2 phosphorylation independently of RAP2A/MAP4K4 and that MST4 degradation and Ezrin dephosphorylation may represent a universal trigger for the biological actions of neratinib.

Global deletion of Optineurin results in altered type I IFN signaling and abnormal bone remodeling in a model of Paget's disease.

Genome-wide association studies (GWAS) have identified Optineurin (OPTN) as genetically linked to Paget's disease of the bone (PDB), a chronic debilitating bone remodeling disorder characterized by localized areas of increased bone resorption and abnormal bone remodeling. However, only ~10% of mouse models with a mutation in Optn develop PDB, thus hindering the mechanistic understanding of the OPTN-PDB axis. Here, we reveal that 100% of aged Optn global knockout (Optn-/-) mice recapitulate the key clinical features observed in PDB patients, including polyostotic osteolytic lesions, mixed-phase lesions, and increased serum levels of alkaline phosphatase (ALP). Differentiation of primary osteoclasts ex vivo revealed that the absence of Optn resulted in an increased osteoclastogenesis. Mechanistically, Optn-deficient osteoclasts displayed a significantly decreased type I interferon (IFN) signature, resulting from both defective production of IFNß and impaired signaling via the IFNα/ßR, which acts as a negative feedback loop for osteoclastogenesis and survival. These data highlight the dual roles of OPTN in the type I IFN response to restrain osteoclast activation and bone resorption, offering a novel therapeutic target for PDB. Therefore, our study describes a novel and essential mouse model for PDB and define a key role for OPTN in osteoclast differentiation.

Regulation of phagolysosomal activity by miR-204 critically influences structure and function of retinal pigment epithelium/retina.

MicroRNA-204 (miR-204) is expressed in pulmonary, renal, mammary and eye tissue, and its reduction can result in multiple diseases including cancer. We first generated miR-204-/- mice to study the impact of miR-204 loss on retinal and retinal pigment epithelium (RPE) structure and function. The RPE is fundamentally important for maintaining the health and integrity of the retinal photoreceptors. miR-204-/- eyes evidenced areas of hyper-autofluorescence and defective photoreceptor digestion, along with increased microglia migration to the RPE. Migratory Iba1+ microglial cells were localized to the RPE apical surface where they participated in the phagocytosis of photoreceptor outer segments (POSs) and contributed to a persistent build-up of rhodopsin. These structural, molecular and cellular outcomes were accompanied by decreased light-evoked electrical responses from the retina and RPE. In parallel experiments, we suppressed miR-204 expression in primary cultures of human RPE using anti-miR-204. In vitro suppression of miR-204 in human RPE similarly showed abnormal POS clearance and altered expression of autophagy-related proteins and Rab22a, a regulator of endosome maturation. Together, these in vitro and in vivo experiments suggest that the normally high levels of miR-204 in RPE can mitigate disease onset by preventing generation of oxidative stress and inflammation originating from intracellular accumulation of undigested photoreactive POS lipids. More generally, these results implicate RPE miR-204-mediated regulation of autophagy and endolysosomal interaction as a critical determinant of normal RPE/retina structure and function.

Neratinib inhibits Hippo/YAP signaling, reduces mutant K-RAS expression, and kills pancreatic and blood cancer cells.

Prior studies demonstrated that the irreversible ERBB1/2/4 inhibitor neratinib caused plasma membrane-associated mutant K-RAS to localize in intracellular vesicles, concomitant with its degradation. Herein, we discovered that neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, K-RAS and N-RAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells. Knock down of Beclin1, ATG16L1, Rubicon or cathepsin B significantly lowered the ability of neratinib to reduce ERBB1 and K-RAS expression, and to cause tumor cell death. Knock down of ATM-AMPK suppressed vesicle formation and knock down of cathepsin B-AIF significantly reduced neratinib lethality. PKG phosphorylates K-RAS and HMG CoA reductase inhibitors reduce K-RAS farnesylation both of which remove K-RAS from the plasma membrane, abolishing its activity. Neratinib interacted with the PKG activator sildenafil and the HMG CoA reductase inhibitor atorvastatin to further reduce K-RAS expression, and to further enhance cell killing. Neratinib is also a Ste20 kinase family inhibitor and in carcinoma cells, and hematopoietic cancer cells lacking ERBB1/2/4, it reduced K-RAS expression and the phosphorylation of MST1/3/4/Ezrin by ~ 30%. Neratinib increased LATS1 phosphorylation as well as that of YAP and TAZ also by ~ 30%, caused the majority of YAP to translocate into the cytosol and reduced YAP/TAZ protein levels. Neratinib lethality was enhanced by knock down of YAP. Neratinib, in a Rubicon-dependent fashion, reduced PAK1 phosphorylation and that of its substrate Merlin. Our data demonstrate that neratinib coordinately suppresses both mutant K-RAS and YAP function to kill pancreatic tumor cells.

Satisfacción de usuarias del control prenatal en Montería / Users' satisfaction with prenatal care in Monteria

Resumen Introducción: En los procesos de atención que requieren seguimiento, como el control prenatal, la satisfacción de la usuaria puede contribuir a su resultado. La percepción de la calidad, estimada mediante la satisfacción de las usuarias, aproxima a la medición de la calidad de la atención prenatal. Objetivo: Establecer el nivel de satisfacción con el control prenatal en una institución prestadora de servicios de salud en Montería (Colombia). Metodología: Estudio transversal con componente analítico, utilizando el programa Epidat 3.1. Se estimaron promedios, medianas y proporciones según fuesen las variables cuantitativas o cualitativas. Se exploró la asociación estadística la prueba t de Student para las variables continuas y la prueba x2 para las variables categóricas, y se estimaron los OR (IC 95%) asumiendo como caso la satisfacción con el control prenatal. Resultados: De las 178 mujeres entrevistadas 96% manifestaron sentirse satisfechas con el control prenatal. Recibieron información sobre: la prueba del Virus de Inmunodeficiencia Humana 83,7%, exámenes de laboratorio 85,4%, signos de alarma 93,8%, nutrición 86,5%, lactancia 76,4%, planificación 64%, cuidados del recién nacido 71,9%. Los promedios de edad (22,7 versus 25,1 años) y de semanas de gestación (11,1 versus 14,9 semanas) son menores en las mujeres satisfechas con el control prenatal (valor p<0,001). Otras variables asociadas con la satisfacción en el control prenatal: entrega completa de medicamentos formulados (OR 8,6 IC 95% 1,6-46,2), oportunidad en la entrega de resultados de los exámenes (OR 6 IC 95% 1,2-28,9), e información suministrada sobre los signos de alarma (OR 20,3 IC 95% 3,7-112) y nutrición (OR 5,3 IC 95% 1,16-25,6). Conclusión: Además de la influencia de las variables demográficas y obstétricas y de los atributos de la calidad de la atención, es importante denotar cómo la información y la educación para la salud se asocian con la satisfacción con el control prenatal.

Abstract Introduction: In the care health processes that require follow-up, such as prenatal care, users satisfaction can contribute to its outcome. The perceived quality, estimated through users satisfaction, approximates the measurement of prenatal care quality. Objective: To establish the level of satisfaction among pregnant women with prenatal care in a health institution in Montería (Colombia). Methodology: Cross-sectional study with descriptive and analytical, using the Epidat 3.1 program. Averages, medians and proportions were estimated according to the quantitative or qualitative variables. The statistical association was explored using the Student's t test for the continuous variables and the x2 test for the categorical variables, and OR was estimated (95%CI) assuming as a case the satisfaction with the prenatal control. Results: Of the 178 women interviewed, 96% reported feeling satisfied with antenatal consultation. They received information on: Human Immunodeficiency Virus test 83.7%, laboratory tests 85.4%, obstetric danger signs 93.8%, maternal nutrition 86.5%, breastfeeding 76.4%, family planning 64%, newborn care 71.9%. The average of age (22.7 versus 25.1 years) and gestational age (11.1 versus 14.9 weeks) are lower in women satisfied with antenatal consultation (p<0.001). Other variables associated with satisfaction in antenatal care: complete medication delivery (OR 8.6 95%CI 1.6-46.2), timely laboratory results (OR 6 95%CI 1.2-28.9), and information provided on obstetric danger signs (OR 20.3 95%CI 3.7-112) and maternal nutrition (OR 5.3 95%CI 1.16-25.16). Conclusion: In addition to the influence of demographic and obstetric variables and attributes of quality care health, it is important to note how information and health education are also associated with women's satisfaction with antenatal care.

Coriocarcinoma no gestacional: reporte de un caso / No gestational choriocarcinoma: case report

Resumen ANTECEDENTES: El coriocarcinoma es una neoplasia maligna del epitelio trofoblástico, agresiva pero susceptible de curación, incluso cuando hay metástasis. Puede ser de origen gestacional o no, el primero aparece después de un embarazo molar o aborto. CASO CLÍNICO: Paciente de 14 años, acudió a Urgencias debido a un cuadro de abdomen agudo, con prueba en orina positiva de embarazo; además, una tumoración en la región anexial derecha. En la laparotomía se encontró una tumoración dependiente del anexo derecho, con la cápsula rota, adherida al colon. Un día después de la intervención quirúrgica (por no contar con recursos en la unidad) se tomó la fracción beta de gonadotropina coriónica. El diagnóstico definitivo se estableció con base en el reporte del estudio histopatológico de la pieza quirúrgica. CONCLUSIÓN: El coriocarcinoma es una neoplasia maligna del epitelio trofoblástico, muy agresiva pero susceptible de curación, incluso cuando se detectan metástasis. La incidencia en México de coriocarcinoma gestacional es de 0.133 por cada 100,000 mujeres, mientras que la incidencia del cariocarcinoma no gestacional se desconoce. Se reporta como un tumor muy raro; por ello el diagnóstico preciso y oportuno es difícil en este tipo de tumores; la presentación más habitual es el abdomen agudo.

Abstract BACKGROUND: Choriocarcinoma is a malignant neoplasm of the trophoblastic epithelium, very aggressive but highly curable, even when there is metastasis. It can be of gestational and non-gestational origin, the first one can develop after a pregnancy, abortion or molar pregnancy; in Mexico it is calculated with an incidence of 0.133 per 100,000 women. While the incidence of non-gestational choriocarcinoma is unknown as it is reported as a very rare tumor. The objective is present the first case reported in our hospital, there are few cases reported in Mexico, it is important to know them in order to identify, diagnose and refer them on time. CLINICAL CASE: A 14-year-old patient who presented to the emergency department with a picture of acute abdomen with pregnancy test in positive urine and tumor in the right adnexal region, laparotomy was performed, finding a tumor dependent on the right annex with a broken capsule attached to the colon, one day after the surgical event (due to lack of resources in the unit) beta fraction of chorionic gonadotropin, the definitive diagnosis was made by the histopathological study of the surgical piece. CONCLUSION: Choriocarcinoma is a malignant neoplasm of the trophoblastic epithelium, very aggressive but highly curable, even when metastases are detected. In Mexico, the incidence of gestational choriocarcinoma is 0.133 per 100,000 women, while the incidence of non-gestational cariocarcinoma is unknown, it is reported as a very rare tumor, which is why accurate and early diagnosis is difficult in this type of tumors. , since the most common presentation is an acute abdomen.

Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent.

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.