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Reducing the costs from human-wildlife conflict, mostly borne by marginal rural households, is a priority for conservation. We estimate the mean species-specific cost for households suffering damages from one of 15 major species of wildlife in India. Our data are from a survey of 5,196 households living near 11 wildlife reserves in India, and self-reported annual costs include crop and livestock losses and human casualties (injuries and death). By employing conservative estimates from the literature on the value of a statistical life (VSL), we find that costs from human casualties overwhelm crop and livestock damages for all species associated with fatalities. Farmers experiencing a negative interaction with an elephant over the last year incur damages on average that are 600 and 900 times those incurred by farmers with negative interactions with the next most costly herbivores: the pig and the nilgai. Similarly, farmers experiencing a negative interaction with a tiger over the last year incur damage that is on average 3 times that inflicted by a leopard and 100 times that from a wolf. These cost differences are largely driven by differences in the incidence of human death and casualties. Our estimate of costs fluctuates across reserves, mostly due to a variation of human casualties. Understanding the drivers of human casualties and reducing their incidence are crucial to reducing the costs from human-wildlife conflict.Most of the tales were about animals, for the Jungle was always at their door. The deer and the pig grubbed up their crops, and now and again the tiger carried off a man at twilight, within sight of the village gates. "Tiger! Tiger!" (Rudyard Kipling, The Jungle Book, Collins Classics, 2010).
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Conservación de los Recursos Naturales/economía , Costos y Análisis de Costo , Productos Agrícolas/economía , Ganado/fisiología , Conducta Predatoria/fisiología , Animales , Animales Salvajes , Agricultores , Humanos , Opinión PúblicaRESUMEN
Many organisms rely on oxygen to generate cellular energy (adenosine triphosphate or ATP). During severe hypoxia, the production of ATP decreases, leading to cell damage or death. Conversely, excessive oxygen causes oxidative stress that is equally damaging to cells. To mitigate pathological outcomes, organisms have evolved mechanisms to adapt to fluctuations in oxygen levels. Zebrafish embryos are remarkably hypoxia-tolerant, surviving anoxia (zero oxygen) for hours in a hypometabolic, energy-conserving state. To begin to unravel underlying mechanisms, we analyze here the distribution of the N-myc Downstream Regulated Gene (ndrg) family, ndrg1-4, and their transcriptional response to hypoxia. These genes have been primarily studied in cancer cells and hence little is understood about their normal function and regulation. We show here using in situ hybridization that ndrgs are expressed in metabolically demanding organs of the zebrafish embryo, such as the brain, kidney, and heart. To investigate whether ndrgs are hypoxia-responsive, we exposed embryos to different durations and severity of hypoxia and analyzed transcript levels. We observed that ndrgs are differentially regulated by hypoxia and that ndrg1a has the most robust response, with a ninefold increase following prolonged anoxia. We further show that this treatment resulted in de novo expression of ndrg1a in tissues where the transcript is not observed under normoxic conditions and changes in Ndrg1a protein expression post-reoxygenation. These findings provide an entry point into understanding the role of this conserved gene family in the adaptation of normal cells to hypoxia and reoxygenation.
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Hipoxia de la Célula , Embrión no Mamífero/metabolismo , Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Oxígeno/metabolismo , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Estrés OxidativoRESUMEN
The aim of this study was to synthesize iodine containing polymeric excipients for mucosal treatment of microbial infection exhibiting a prolonged mucosal residence time by forming an adhesive gel on the mucosal surface. In order to achieve this aim, 2-(2 acryloylamino-ethyldisulfanyl)-nicotinic acid (ACENA) was copolymerized with N-vinylpyrrolidone (NVP) to obtain thiolated polyvinylpyrrolidone (PVP) for complexation with iodine. The average molecular mass of different thiolated PVP variants was determined by size exclusion chromatography. The structure of thiolated PVP was confirmed by 1H NMR. Thiolated PVP variants were characterized for thiol content, cytotoxicity, iodine loading capacity, rheological behavior, and adhesion time on mucosa. The highest achieved degree of thiolation was 610 ± 43 µmol/g, and the maximum recorded iodine loading was 949 ± 31 µmol/g of polymer. Thiolated PVP variants (0.5% m/v) showed no toxicity after incubation on Caco-2 cells for the period of 3 and 24 h, respectively. Thiolated PVP and thiolated PVP-iodine complexes exhibited a 5.4- and 4.4-fold increased dynamic viscosity in porcine mucus in comparison to PVP and PVP-iodine complex, respectively. Compared to PVP and PVP-iodine complex thiol-functionalized PVP and PVP-iodine complexes demonstrated significantly prolonged attachment to mucosal surface over a period of 3 h. Thiol functionalized PVP proved to be a promising novel excipient for complexation with iodine and to exhibit strongly improved mucoadhesive properties.
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Adhesivos/farmacología , Antiinfecciosos Locales/farmacología , Excipientes/farmacología , Povidona Yodada/farmacología , Compuestos de Sulfhidrilo/farmacología , Adhesivos/síntesis química , Animales , Antiinfecciosos Locales/síntesis química , Células CACO-2 , Composición de Medicamentos/métodos , Excipientes/síntesis química , Glicoproteínas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Povidona Yodada/síntesis química , Compuestos de Sulfhidrilo/síntesis química , PorcinosRESUMEN
BACKGROUND: Prosthetic hip-associated cobalt toxicity (PHACT) is an uncommon, but potentially devastating, complication for patients with metal-on-metal hip implants (MoMs). Clinical management of PHACT is poorly defined, with primary intervention being MoM explant followed by chelation therapy. Therapeutic plasma exchange (TPE) in cobalt toxicity has not been previously described. Given that cobalt is predominantly albumin bound, it should theoretically be removed by TPE. Here we report a case of PHACT and our experience using TPE to lower plasma cobalt levels. CASE REPORT: A 61-year-old woman developed deafness, blindness, ambulatory dysfunction, and endocrinopathies after MoM implant. Cobalt levels on admission were greater than 1500 µg/L. In an attempt to rapidly lower cobalt levels before MoM explant, hemodialysis and TPE were performed. Hemodialysis removed negligible amounts of cobalt. One session of TPE temporarily removed approximately two-thirds of measurable cobalt, but levels rebounded to pre-TPE values after 8 hours. It was only after MoM removal that cobalt levels plateaued below 300 µg/L and clinical symptoms improved. DISCUSSION: TPE removed cobalt from a PHACT patient, but a durable decrease in cobalt was only achieved after MoM explant. These findings are comparable to reports where chelation was employed in PHACT patients before MoM explant. The observed rebound phenomenon is likely from rapid equilibration between the immense extravascular tissue source (the MoM) and the intravascular compartment. CONCLUSION: TPE may serve as adjunctive therapy for PHACT patients whose cobalt levels remain high after explant, especially in patients with renal failure, in whom chelation is contraindicated.
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Cobalto/toxicidad , Prótesis de Cadera/efectos adversos , Intercambio Plasmático/métodos , Artroplastia de Reemplazo de Cadera/efectos adversos , Terapia por Quelación , Femenino , Humanos , Persona de Mediana EdadRESUMEN
BACKGROUND: Regulations governing pretransfusion testing allow specimen expiration to be extended past 3 days before the transfusion if a patient has not been transfused or pregnant in the preceding 3 months. Our hospital allows extension of the expiration of a presurgical specimen to 28 days if 1) the patient has neither been transfused nor pregnant in the past 3 months, 2) the patient does not have an antibody history, and 3) the current antibody screen (ABSC) is negative. Patients not meeting Criteria 2 and 3 are required to have specimens redrawn on the day of surgery (DOS). We evaluated the necessity of this policy. STUDY DESIGN AND METHODS: From October 2009 to September 2010, there were 132 patients who did not meet the above criteria for specimen extension. Equivalent tests were performed on preadmission testing (PAT) and DOS specimens, and the results were compared. RESULTS: The majority (113, 86%) of the samples redrawn on the DOS showed no change in antibody serology upon reinvestigation. Of the remaining patients, DOS specimens did not identify any new antibodies or change in blood product choices. CONCLUSION: Of the PAT specimens rejected for antibody history or positive ABSC, none had new significant serologic findings on DOS. Based on these results, requiring a repeat specimen on the DOS may not be clinically necessary. Our facility changed the PAT policy to extend specimen acceptability to patients with red blood cell antibody history or positive ABSC at time of PAT. A 6-month follow-up period showed that this practice is safe.
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Recolección de Muestras de Sangre/normas , Política Organizacional , Admisión del Paciente , Seguridad del Paciente/legislación & jurisprudencia , Seguridad del Paciente/normas , Tipificación y Pruebas Cruzadas Sanguíneas/normas , Conservación de la Sangre/normas , Transfusión Sanguínea/legislación & jurisprudencia , Transfusión Sanguínea/normas , Femenino , Estudios de Seguimiento , Hospitales/normas , Humanos , Isoanticuerpos/análisis , Isoanticuerpos/sangre , Admisión del Paciente/legislación & jurisprudencia , Admisión del Paciente/normas , Seguridad del Paciente/estadística & datos numéricos , Hemorragia Posoperatoria/epidemiología , Hemorragia Posoperatoria/terapia , Embarazo , Pruebas Serológicas , Factores de Tiempo , Reacción a la Transfusión , Estados Unidos/epidemiología , United States Food and Drug Administration/legislación & jurisprudenciaRESUMEN
BACKGROUND: Our blood bank is part of a large academic institution with an active sickle cell anemia program. We provide sickle patients with blood phenotypically matched for C/c, E/e, and K antigens. Since licensed reagents are available for phenotyping C/c, E/e, and K on an automated blood analyzer, we decided to evaluate whether establishing our own inventory of blood negative for those antigens would result in cost savings and decreased turnaround time (TAT). STUDY DESIGN AND METHODS: Antigen typing of blood units for C/c, E/e, and K was validated. From March 1, 2012, to August 31, 2012, a total of 1033 units from our own donor center and from our suppliers were phenotyped. We compared direct cost savings and TAT for blood availability with historical data before we began phenotyping. RESULTS: Thirty-eight percent of typed antigen-negative (AG-) units were transfused to sickle patients. An additional 35% were transfused to nonsickle patients needing AG- blood. Twenty-one percent were used by patients without antibodies to prevent outdating. The remaining 6% had not yet been transfused by the end of the study period. From March 1, 2011, to August 31, 2011, we spent almost $200,000 on obtaining AG- blood. In the 6 months since we started antigen typing, we have saved approximately $110,000, the majority of which resulted from AG- blood provided to sickle patients. In addition, TAT for AG- units from our inventory significantly improved to 1 to 2 hours versus approximately 6 hours when obtained from our suppliers. CONCLUSION: Establishing an AG- inventory in a hospital-based blood bank is cost-effective and time-efficient.
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Anemia de Células Falciformes/terapia , Almacenamiento de Sangre/métodos , Bancos de Sangre/economía , Antígenos de Grupos Sanguíneos/inmunología , Eritrocitos/inmunología , Centros Médicos Académicos/economía , Tipificación y Pruebas Cruzadas Sanguíneas/economía , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Transfusión Sanguínea , Ahorro de Costo , HumanosRESUMEN
Due to its versatility in formulation and manufacturing, self-emulsifying drug delivery systems (SEDDS) can be used to design parenteral formulations. Therefore, it is necessary to understand the effects of excipients on the behavior of SEDDS formulations upon parenteral administration, particularly their interactions with blood plasma and cell membranes. In this study, we prepared three neutrally charged SEDDS formulations composed of medium-chain triglycerides as the oil phase, polyoxyl-35 castor oil (EL35) and polyethylene glycol (15)-hydroxystearate (HS15) as the nonionic surfactants, medium-chain mono- and diglycerides as the co-surfactant, and propylene glycol as the co-solvent. The cationic surfactant, didodecyldimethylammonium bromide (DDA), and the anionic surfactant, sodium deoxycholate (DEO), were added to the neutral SEDDS preconcentrates to obtain cationic and anionic SEDDS, respectively. SEDDS were incubated with human blood plasma and recovered by size exclusion chromatography. Data showed that SEDDS emulsion droplets can bind plasma protein to different extents depending on their surface charge and surfactant used. At pH 7.4, the least protein binding was observed with anionic SEDDS. Positive charges increased protein binding. SEDDS stabilized by HS15 can adsorb more plasma protein and induce more plasma membrane disruption activity than SEDDS stabilized by EL35. These effects were more pronounced with the HS15 + DDA combination. The addition of DDA and DEO to SEDDS increased plasma membrane disruption (PMD) activities, and DDA (1% w/w) was more active than DEO (2% w/w). PMD activities of SEDDS were concentration-dependent and vanished at appropriate dilution ratios.
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Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for treating neurodegenerative diseases. In this study, we present an efficient method for reprogramming retinal glial cells into neurons. By suppressing Notch signaling by disrupting either Rbpj or Notch1/2, we induced mature Müller glial cells to reprogram into bipolar- and amacrine-like neurons. We demonstrate that Rbpj directly activates both Notch effector genes and genes specific to mature Müller glia while indirectly repressing expression of neurogenic basic helix-loop-helix (bHLH) factors. Combined loss of function of Rbpj and Nfia/b/x resulted in conversion of nearly all Müller glia to neurons. Last, inducing Müller glial proliferation by overexpression of dominant-active Yap promotes neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia. These findings demonstrate that Notch signaling and NFI factors act in parallel to inhibit neurogenic competence in mammalian Müller glia and help clarify potential strategies for regenerative therapies aimed at treating retinal dystrophies.
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Reprogramación Celular , Células Ependimogliales , Factores de Transcripción NFI , Neuroglía , Neuronas , Receptores Notch , Retina , Transducción de Señal , Animales , Factores de Transcripción NFI/metabolismo , Factores de Transcripción NFI/genética , Ratones , Retina/metabolismo , Retina/citología , Células Ependimogliales/metabolismo , Células Ependimogliales/citología , Neuroglía/metabolismo , Receptores Notch/metabolismo , Neuronas/metabolismo , Neuronas/citología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Neurogénesis , Proteínas Señalizadoras YAP/metabolismo , Proliferación CelularRESUMEN
Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for neurodegenerative diseases. Despite successful applications in vitro , in vivo implementation has been hampered by low efficiency. In this study, we present a highly efficient strategy for reprogramming retinal glial cells into neurons by simultaneously inhibiting key negative regulators. By suppressing Notch signaling through the removal of its central mediator Rbpj, we induced mature Müller glial cells to reprogram into bipolar and amacrine neurons in uninjured adult mouse retinas, and observed that this effect was further enhanced by retinal injury. We found that specific loss of function of Notch1 and Notch2 receptors in Müller glia mimicked the effect of Rbpj deletion on Müller glia-derived neurogenesis. Integrated analysis of multiome (scRNA- and scATAC-seq) and CUT&Tag data revealed that Rbpj directly activates Notch effector genes and genes specific to mature Müller glia while also indirectly represses the expression of neurogenic bHLH factors. Furthermore, we found that combined loss of function of Rbpj and Nfia/b/x resulted in a robust conversion of nearly all Müller glia to neurons. Finally, we demonstrated that inducing Müller glial proliferation by AAV (adeno-associated virus)-mediated overexpression of dominant- active Yap supports efficient levels of Müller glia-derived neurogenesis in both Rbpj - and Nfia/b/x/Rbpj - deficient Müller glia. These findings demonstrate that, much like in zebrafish, Notch signaling actively represses neurogenic competence in mammalian Müller glia, and suggest that inhibition of Notch signaling and Nfia/b/x in combination with overexpression of activated Yap could serve as an effective component of regenerative therapies for degenerative retinal diseases.
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Chitosan (Ch) and different Ch derivatives have been applied in tissue engineering (TE) because of their biocompatibility, favored mechanical properties, and cost-effectiveness. Most of them, however, lack cell adhesive properties that are crucial for TE. In this study, we aimed to design an S-protected thiolated Ch derivative exhibiting high cell adhesive properties serving as a scaffold for TE. 3-((2-Acetamido-3-methoxy-3-oxopropyl)dithio) propanoic acid was covalently attached to Ch via a carbodiimide-mediated reaction. Low-, medium-, and high-modified Chs (Ch-SS-1, Ch-SS-2, and Ch-SS-3) with 54, 107 and 140 µmol of ligand per gram of polymer, respectively, were tested. In parallel, three thiolated Chs, namely Ch-SH-1, Ch-SH-2, and Ch-SH-3, were prepared by conjugating N-acetyl cysteine to Ch at the same degree of modification to compare the effectiveness of disulfide versus thiol modification on cell adhesion. Ch-SS-1 showed better cell adhesion capability than Ch-SS-2 and Ch-SS-3. This can be explained by the more lipophilic surfaces of Ch-SS as a higher modification was made. Although Ch-SH-1, Ch-SH-2, and Ch-SH-3 were shown to be good substrates for cell adhesion, growth, and proliferation, Ch-SS polymers were superior to Ch-SH polymers in the formation of 3D cell cultures. Cryogels structured by Ch-SS-1 (SSg) resulted in homogeneous scaffolds with tunable pore size and mechanical properties by changing the mass ratio between Ch-SS-1 and heparin used as a cross-linker. SSg scaffolds possessing interconnected microporous structures showed good cell migration, adhesion, and proliferation. Therefore, Ch-SS can be used to construct tunable cryogel scaffolds that are suitable for 3D cell culture and TE.
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Quitosano , Materiales Biocompatibles/farmacología , Ingeniería de Tejidos , Acetilcisteína , Carbodiimidas , CriogelesRESUMEN
Direct reprogramming of retinal Müller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Müller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Müller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.
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HYPOTHESIS: The aim of this study was the development of nanostructured lipid carriers (NLCs) decorated with a polycationic cell-penetrating peptide (CPP). A coating with polyphosphates (PP) enables charge conversion at target cells being triggered by the membrane bound enzyme intestinal alkaline phosphatase (IAP). EXPERIMENTS: The CPP, stearyl-nona-L-arginine (R9SA) was obtained by solid phase synthesis. Formed nanocarriers were characterized regarding size, polydispersity index, zeta potential and charge conversion in the presence of IAP and on Caco-2 cells. The BCS class IV drug saquinavir (SQV) was loaded into NLCs in different concentrations. Mucus diffusion ability of the NLCs was evaluated by the rotating tube method. Furthermore, cellular uptake was evaluated on Caco-2 cells and endosomal escape properties were investigated using erythrocytes. FINDINGS: All NLCs were obtained in a size range between 146 nm and 152 nm and a polydispersity index of 0.2. Incubation of PP coated PP-R9SA-NLCs with IAP led to a charge conversion from -41.8 mV to 6.4 mV (Δ48.2 mV). After four hours of incubation with IAP, phosphate release reached a plateau, indicating a faster polyphosphate cleavage than on Caco-2. Drug load and encapsulation efficiency of SQV was obtained up to 80.6% and 46.5 µg/mg. Mucus diffusion was increasing in the following rank order: R9SA-NLCs < blank NLCs < PP-R9SA-NLCs. R9SA-NLCs and PP-R9SA-NLCs increased the cellular uptake 15.6- and 13.2-fold, respectively, compared to the control NLCs. Erythrocytes interaction study revealed enhanced endosomal escape properties for R9SA-NLCs and PP-R9SA-NLCs when incubated with IAP.
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Péptidos de Penetración Celular , Nanoestructuras , Fosfatasa Alcalina , Células CACO-2 , Portadores de Fármacos/química , Humanos , Lípidos/química , Nanoestructuras/química , Tamaño de la Partícula , Polifosfatos , Saquinavir/químicaRESUMEN
AIM: Evaluation of different polyhydroxy surfaces in SEDDS to overcome the limitations associated with conventional polyethylene glycol (PEG)-based SEDDS surfaces for intracellular drug delivery. METHODS: Anionic, cationic and non-ionic polyglycerol- (PG-) and alkylpolyglucoside- (APG-) surfactant based SEDDS were developed and compared to conventional PEG-SEDDS. Particular emphasis was placed on the impact of SEDDS surface decoration on size and zeta potential, drug loading and protective effect, mucus diffusion, SEDDS-cell interaction and intracellular delivery of the model drug curcumin. RESULTS: After self-emulsification, SEDDS droplets sizes were within the range of 35-190 nm. SEDDS formulated with high amounts of long PEG-chain surfactants (>10 monomers) a charge-shielding effect was observed. Replacing PEG-surfactants with PG- and an APG-surfactant did not detrimentally affect SEDDS self-emulsification, payloads or the protection of incorporated curcumin towards oxidation. PG- and APG-SEDDS bearing multiple hydroxy functions on the surface demonstrated mucus permeation comparable to PEG-SEDDS. Steric hinderance and charge-shielding of PEG-SEDDS surface substantially reduced cellular uptake up to 50-fold and impeded endosomal escape, yielding in a 20-fold higher association of PEG-SEDDS with lysosomes. In contrast, polyhydroxy-surfaces on SEDDS promoted pronounced cellular internalisation and no lysosomal co-localisation was observed. This improved uptake resulted in an over 3-fold higher inhibition of tumor cell proliferation after cytosolic curcumin delivery. CONCLUSION: The replacement of PEG-surfactants by surfactants with polyhydroxy head groups in SEDDS is a promising approach to overcome the limitations for intracellular drug delivery associated with conventional PEGylated SEDDS surfaces.
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Curcumina , Tensoactivos , Sistemas de Liberación de Medicamentos/métodos , Emulsionantes/metabolismo , Emulsiones , Excipientes , Polietilenglicoles , SolubilidadRESUMEN
AIM: To prepare new polycationic pullulan derivatives exhibiting highly mucoadhesive and sustained drug release properties. METHODS: Hydroxy groups of pullulan were activated with mesyl chloride followed by conjugation with low-molecular weight polyamines. Pullulan-tris(2-aminoethyl)amine (Pul-TAEA) and pullulan-polyethyleneimine (Pul-PEI) were evaluated regarding swelling behaviour, mucoadhesive properties and potential to control drug release. RESULTS: Pul-TAEA and Pul-PEI exhibited excellent swelling properties at pH 6.8 showing 240- and 370-fold increase in weight. Compared to unmodified pullulan, Pul-TAEA and Pul-PEI displayed 5- and 13.3-fold increased dynamic viscosity in mucus. Mucoadhesion studies of Pul-TAEA and Pul-PEI on intestinal mucosa showed a 6- and 37.8-fold increase in tensile strength, and a 72- and 120-fold increase in mucoadhesion time compared to unmodified pullulan, respectively. Due to additional ionic interactions between cationic groups on polyaminated pullulans and an anionic model drug, a sustained drug release was achieved. CONCLUSIONS: Polyaminated pullulans are promising novel mucoadhesive excipients for mucosal drug delivery.
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Sistemas de Liberación de Medicamentos , Etilenodiaminas , Glucanos , Mucosa Intestinal/química , Polietileneimina , Adhesividad , Animales , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos , Etilenodiaminas/administración & dosificación , Etilenodiaminas/química , Glucanos/administración & dosificación , Glucanos/química , Glicósido Hidrolasas/química , Humanos , Moco/química , Polietileneimina/administración & dosificación , Polietileneimina/química , Reología , Porcinos , Resistencia a la Tracción , ViscosidadRESUMEN
Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor (NARF) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.
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Neoplasias de la Mama , Factor 1 Inducible por Hipoxia , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Histonas/metabolismo , Hipoxia/genética , Hipoxia/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/fisiología , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
AIM: This study aimed to develop phosphatase-responsive ζ potential converting nanocarriers utilizing polyphosphate-coated cell-penetrating peptide (CPP)-decorated nanoemulsions (NEs) as a novel gene delivery system to retinal cells. METHODS: Poly-l-lysine (PLL) was first conjugated with oleylamine (OA) only at its carboxylic end to form the amphiphilic PLL-oleylamine (PLOA) conjugate. Afterward, NEs were loaded with PLOA prior to being coated with tripolyphosphate (TPP) to generate PLOA/TPP NEs. A plasmid containing a reporter gene for green fluorescent protein plasmid (pGFP) was complexed with cationic surfactants forming hydrophobic ion pairs that were loaded in the oily core of NEs. Phosphate removal, ζ potential conversion, and cytotoxicity of the system were evaluated. Cellular uptake and transfection efficiency were investigated in 661W photoreceptor-like cells via microscopic analysis, fluorescence spectroscopy, and flow cytometry. RESULTS: Dephosphorylation of PLOA/TPP NEs triggered by alkaline phosphatase (ALP) resulted in the exposure of positive amine groups on the surface of NE droplets and a notable conversion of the ζ potential from -22.4 to +8.5 mV. Cellular uptake of PLOA/TPP NEs performed on 661W photoreceptor-like cells showed a 3-fold increase compared to control NEs. Furthermore, PLOA/TPP NEs also showed low cytotoxicity and high transfection efficacy with â¼50% of cells transfected. CONCLUSIONS: Polyphosphate-coated CPP-decorated NEs triggered by ALP could be a promising nanosystem to efficiently deliver drugs and genetic materials to photoreceptor-like cells and other retinal cells for potential treatments of retinal diseases.
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Péptidos de Penetración Celular , Nanopartículas , Fosfatasa Alcalina , Aminas , Sistemas de Liberación de Medicamentos/métodos , Emulsiones/química , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Nanopartículas/química , Polilisina , Polifosfatos/química , Tensoactivos/químicaRESUMEN
AIM: The aim of this study was to form hydrophobic ion-pairs of proteinase with cationic surfactants and to incorporate them into self-emulsifying drug delivery systems (SEDDS) to improve their mucus permeating properties. METHODS: Proteinase was ion-paired with benzalkonium chloride (BAK), hexadecylpyridinium chloride (HDP), alkyltrimethylammonium bromide (ATA) and hexadecyltrimethylammonium bromide (HDT) at pH 8.5-9.0, and subsequently incorporated into SEDDS consisting of Cremophor EL, propylene glycol, and Capmul 808-G (40/20/40). Mucus permeation of SEDDS containing proteinase complexes was evaluated via rotating tube technique and cell-free Transwell® insert system. Additionally, enzymatic activity of proteinase complexes as well as their potential cytotoxicity was evaluated. RESULTS: Among all tested hydrophobic ion-pairs, proteinase/BAK showed highest potential. Mucus diffusion of SEDDS containing proteinase/BAK complex yielded in 2.3-fold and 2.5-fold higher mucus permeability with respect to blank SEDDS at Transwell® insert system and rotating tube technique, respectively. Furthermore, proteinase/BAK complex maintained the highest enzymatic activity of 50.5 ± 5.6% compared to free proteinase. At a SEDDS concentration as low as 0.006% cell viability was just 80%. The addition of proteinase complexes to SEDDS increased cytotoxicity on Caco-2 cells in a concentration-dependent manner. CONCLUSION: SEDDS loaded with proteinase/BAK complexes are promising nanocarriers because of enhanced mucus permeating properties.
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Emulsionantes , Expectorantes , Células CACO-2 , Sistemas de Liberación de Medicamentos , Emulsiones , Humanos , Péptido HidrolasasRESUMEN
In recent decades, three generations of thiomers have been developed with the main purpose of obtaining enhanced interactions with mucosal tissues. Therefore, many different types of thiolated ligands have been generated and attached to polymeric backbones. The aim of this study was to synthesize all three generations of thiomers and to directly compare their properties regarding mucus penetration and mucoadhesion. Starting from pectin, the unprotected thiomer pectin-cysteine (Pec-Cys), the preactivated S-protected thiomer pectin-cysteine-mercaptonicotinic acid (Pec-Cys-MNA) and the less reactive S-protected thiomer pectin-cysteine-glutathione (Pec-Cys-GSH) were synthesized and characterised by FT-IR, NMR, and colorimetric studies. The polymers were evaluated regarding their cytotoxicity, swelling behaviour, viscosity after mixing with mucus, mucus diffusion, penetration into mucosa, and mucoadhesion. The amount of the three ligands (Cys, Cys-MNA and Cys-GSH) bound to the polymer was determined to be in the range of 193-196 µmol/g. All polymers showed no cytotoxicity. Viscosity of the mixture of Pec-Cys-MNA and Pec-Cys-GSH with mucus increased 21.5- and 26.7-fold, respectively, compared to the unmodified polymer within 3 hours. Swelling, mucoadhesion, interpenetration and mucus diffusion were increased in the following rank order: Pec-Cys < Pec-Cys-MNA < Pec-Cys-GSH. Results of mucoadhesion study indicated a 7.4 and 8.1-fold increase of Pec-Cys-MNA and Pec-Cys-GSH, respectively, compared to the unmodified polymer. As the less reactive S-protected thiomer exhibited higher mucoadhesive properties than the other thiomers, this study provides evidence for the superior mucoadhesion of 3rd generation thiomers. STATEMENT OF SIGNIFICANCE: Three generations of thiolated polymers have been developed bearing different types of thiol ligands with the main purpose of enhancing mucus interactions. In this study, all generations were synthesized on the polymeric backbone of pectin for the first time to directly compare their mucus penetrating and mucoadhesive properties. 1st generation exhibited covalently bound L-cysteine moieties. For 2nd generation, thiols of cysteines were S-protected with 2-mercaptonicotinic acid (MNA), resulting in high reactive disulfide bonds. 3rd generation was synthesized by a thiol/disulfide exchange of glutathione with MNA, producing a less reactive disulfide bond. Mucus penetrating and mucoadhesive properties were found to be increased as follows: 1st generation < 2nd generation < 3rd generation. According to these results, the thiomer of 3rd generation represents a promising excipient with strong mucoadhesion.
Asunto(s)
Pectinas , Compuestos de Sulfhidrilo , Células CACO-2 , Cisteína , Sistemas de Liberación de Medicamentos , Humanos , Polímeros , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
AIM: It was the aim of this study to develop a zeta potential changing drug delivery system by decorating lipid-based nanocarriers with a polycationic cell penetrating peptide (CPP) and subsequently masking these cationic substructures with polyphosphates. METHODS: In order to anchor the CPP poly-l-lysine (PLL) on the surface of the oily droplets of an o/w nanoemulsion, stearic acid was covalently attached to the peptide. The resulting CPP-decorated oily droplets were coated with phytic acid and tripolyphosphate. The elimination of these polyphosphates due to cleavage by alkaline phosphatase was monitored by the release of monophosphate from the surface of the nanocarriers, by the change in zeta potential and by cellular uptake studies on Caco-2 cells. RESULTS: Polyphosphate coated PLL-decorated nanocarriers exhibited a pronounced conversion of zeta potential from -14.1 mV to +4.2 mV in case of tripolyphosphate coated nanocarriers and from -9.9 mV to -2.6 mV in case of phytic acid coated nanocarriers. The cellular uptake on Caco-2 cells of the polyphosphate coated nanocarriers was 4-fold improved compared to the control nanocarriers. Furthermore, confocal images showed that the majority of nanodroplets distributed in cytoplasm not being internalized into lysosomes. CONCLUSION: Polyphosphate coating of CPP-decorated nanocarriers seems to be a promising and simple strategy to overcome the polycation dilemma.
Asunto(s)
Sistemas de Liberación de Medicamentos , Polifosfatos , Células CACO-2 , Humanos , PolielectrolitosRESUMEN
Hypoxia-inducible factors (HIFs) activate transcription of target genes by recruiting coactivators and chromatin-modifying enzymes. Peptidylarginine deiminase 4 (PADI4) catalyzes the deimination of histone arginine residues to citrulline. Here, we demonstrate that PADI4 expression is induced by hypoxia in a HIF-dependent manner in breast cancer and hepatocellular carcinoma cells. PADI4, in turn, is recruited by HIFs to hypoxia response elements (HREs) and is required for HIF target gene transcription. Hypoxia induces histone citrullination at HREs that is PADI4 and HIF dependent. RNA sequencing revealed that almost all HIF target genes in breast cancer cells are PADI4 dependent. PADI4 is required for breast and liver tumor growth and angiogenesis in mice. PADI4 expression is correlated with HIF-1α expression and vascularization in human breast cancer biopsies. Thus, HIF-dependent recruitment of PADI4 to target genes and local histone citrullination are required for transcriptional responses to hypoxia.