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Diabetic foot ulcers (DFUs) present significant challenges due to their associated amputation rates, mortality, treatment complexity and excessive costs. Our earlier work introduced a wound surgical integrated treatment (WSIT) for DFUs, yielding promising outcomes. This study focuses on a specific WSIT protocol employing antibiotic-loaded bone cement (ALBC) in the first Stage, and free vastus lateralis muscle-sparing (VLMS) flaps and split-thickness skin grafts (STSGs) in the second stage to repair non-weight-bearing DFUs. From July 2021 to July 2023, seven DFU patients (aged 47-71 years) underwent this treatment. Demographic data, hospital stay and repair surgery times were collected. Histological and immunohistochemical analyses assessed angiogenesis, collagen deposition and inflammation. SF-36 questionnaire measured pre- and postoperative quality of life. Preoperative ultrasound Doppler showed that the peak blood flow velocity of the recipient area artery was significantly >30 cm/s (38.6 ± 6.8 cm/s) in all patients. Muscle flap sizes varied from 8 × 3.5 × 1 to 18 × 6 × 2 cm. The operation time of the repair surgery was 156.9 ± 15.08 minutes, and the hospital stay was 18.9 ± 3.3 days. Histological analysis proved that covering DFUs with ALBC induced membrane formation and increased collagen, neovascularization and M2 macrophages fraction while reducing M1 macrophages one. All grafts survived without amputation during a 7- to 24-month follow-up, during which SF-36 scores significantly improved. A combination of ALBC with free VLMS flaps and STSGs proved to be safe and effective for reconstructing non-weight-bearing DFUs. It rapidly controlled infection, enhanced life quality and foot function, and reduced hospitalization time. We advocate integrating this strategy into DFU treatment plans.
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Antibacterianos , Cementos para Huesos , Pie Diabético , Trasplante de Piel , Humanos , Pie Diabético/cirugía , Persona de Mediana Edad , Masculino , Anciano , Femenino , Trasplante de Piel/métodos , Antibacterianos/uso terapéutico , Antibacterianos/administración & dosificación , Cementos para Huesos/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Procedimientos de Cirugía Plástica/métodos , Colgajos Tisulares Libres , Músculo CuádricepsRESUMEN
The rapid advancement of nanomedicine and nanoparticle (NP) materials presents novel solutions potentially capable of revolutionizing health care by improving efficacy, bioavailability, drug targeting, and safety. NPs are intriguing when considering medical applications because of their essential and unique qualities, including a significantly higher surface to mass ratio, quantum properties, and the potential to adsorb and transport drugs and other compounds. However, NPs must overcome or navigate several biological barriers of the human body to successfully deliver drugs at precise locations. Engineering the drug carrier biointerface can help overcome the main biological barriers and optimize the drug delivery in a more personalized manner. This review discusses the significant heterogeneous biological delivery barriers and how biointerface engineering can promote drug carriers to prevail over hurdles and navigate in a more personalized manner, thus ushering in the era of Precision Medicine. We also summarize the nanomedicines' current advantages and disadvantages in drug administration, from natural/synthetic sources to clinical applications. Additionally, we explore the innovative NP designs used in both non-personalized and customized applications as well as how they can attain a precise therapeutic strategy.
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Sistemas de Liberación de Medicamentos , Nanopartículas , Portadores de Fármacos , Humanos , Nanomedicina , Nanopartículas/uso terapéutico , Medicina de PrecisiónRESUMEN
Fibrillar aggregates and soluble oligomers of both Amyloid-ß peptides (Aßs) and hyperphosphorylated Tau proteins (p-Tau-es), as well as a chronic neuroinflammation are the main drivers causing progressive neuronal losses and dementia in Alzheimer's disease (AD). However, the underlying pathogenetic mechanisms are still much disputed. Several endogenous neurotoxic ligands, including Aßs, and/or p-Tau-es activate innate immunity-related danger-sensing/pattern recognition receptors (PPRs) thereby advancing AD's neuroinflammation and progression. The major PRR families involved include scavenger, Toll-like, NOD-like, AIM2-like, RIG-like, and CLEC-2 receptors, plus the calcium-sensing receptor (CaSR). This quite intricate picture stresses the need to identify the pathogenetically topmost Aß-activated PRR, whose signaling would trigger AD's three main drivers and their intra-brain spread. In theory, the candidate might belong to any PRR family. However, results of preclinical studies using in vitro nontumorigenic human cortical neurons and astrocytes and in vivo AD-model animals have started converging on the CaSR as the pathogenetically upmost PRR candidate. In fact, the CaSR binds both Ca2+ and Aßs and promotes the spread of both Ca2+ dyshomeostasis and AD's three main drivers, causing a progressive neurons' death. Since CaSR's negative allosteric modulators block all these effects, CaSR's candidacy for topmost pathogenetic PRR has assumed a growing therapeutic potential worth clinical testing.
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Enfermedad de Alzheimer/metabolismo , Encéfalo/patología , Inflamación/patología , Receptores de Reconocimiento de Patrones/metabolismo , Animales , Humanos , Inflamasomas/metabolismo , Neuroglía/metabolismo , Neuroglía/patologíaAsunto(s)
Dermatología , Envejecimiento de la Piel , Humanos , Envejecimiento de la Piel/fisiología , Piel , AnimalesRESUMEN
Mitochondrial disorders are heterogeneous multisystemic disorders due to impaired oxidative phosphorylation causing defective mitochondrial energy production. Common histological hallmarks of mitochondrial disorders are RRFs (ragged red fibres), muscle fibres with abnormal focal accumulations of mitochondria. In contrast with the growing understanding of the genetic basis of mitochondrial disorders, the fate of phenotypically affected muscle fibres remains largely unknown. We investigated PCD (programmed cell death) in muscle of 17 patients with mitochondrial respiratory chain dysfunction. We documented that in affected muscle fibres, nuclear chromatin is condensed in lumpy irregular masses and cytochrome c is released into the cytosol to activate, along with Apaf-1 (apoptotic protease-activating factor 1), caspase 9 that, in turn, activates effector caspase 3, caspase 6, and caspase 7, suggesting the execution of the intrinsic apoptotic pathway. Whereas active caspase 3 underwent nuclear translocation, AIF (apoptosis-inducing factor) mainly stayed within mitochondria, into which an up-regulated Bax is relocated. The significant increase in caspase 2, caspase 3 and caspase 6 activity strongly suggest that the cell death programme is caspase-dependent and the activation of caspase 2 together with PUMA (p53 up-regulated modulator of apoptosis) up-regulation point to a role for oxidative stress in triggering the intrinsic pathway. Concurrently, in muscle of patients, the number of satellite cells was significantly increased and myonuclei were detected at different stages of myogenic differentiation, indicating that a reparative programme is ongoing in muscle of patients with mitochondrial disorders. Together, these data suggest that, in patients with mitochondrial disorders, affected muscle fibres are trapped in a mitochondria-regulated caspase-dependent PCD while repairing events take place.
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Apoptosis , Caspasas/metabolismo , Enfermedades Mitocondriales/fisiopatología , Fibras Musculares Esqueléticas/fisiología , Estudios de Casos y Controles , Humanos , Enfermedades Mitocondriales/enzimologíaRESUMEN
The "amyloid-ß (Aß) hypothesis" posits that accumulating Aß peptides (Aßs) produced by neurons cause Alzheimer's disease (AD). However, the Aßs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aß25-35, an Aß42 proxy, evokes a surplus endogenous Aß42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAß25-35 and soluble (s)Aß25-35 on Aß42 and Aß40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aßs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAß25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aß42, while Aß40 also accrued but its secretion was unchanged. Accordingly, secreted Aß42/Aß40 ratio values rose for astrocytes and neurons. While slightly enhancing Aß40 secretion by fAß25-35-treated astrocytes, NPS 2143 specifically suppressed the fAß25-35-elicited surges of endogenous Aß42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aß42/Aß40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/ß-secretase and γ-secretase activity in fAß25-35-treated astrocytes. Compared to fAß25-35, sAß25-35 also stimulated Aß42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aß/CaSR-induced surplus secretion of endogenous Aß42 and hence further vicious cycles of Aß self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Astrocitos/efectos de los fármacos , Naftalenos/farmacología , Neuronas/efectos de los fármacos , Receptores Sensibles al Calcio/antagonistas & inhibidores , Adulto , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/fisiología , Astrocitos/metabolismo , Biopterinas/análogos & derivados , Biopterinas/farmacología , Células Cultivadas , Humanos , Naftalenos/uso terapéutico , Neuronas/metabolismoRESUMEN
The in situ Proximity Ligation Assay (PLA) is suited for visualizing protein-protein interactions and post-translational protein modifications in both tissue sections and in vitro cell cultures. Accurate identification and quantification of protein-protein interactions are critical for in vitro cell analysis, especially when studying the dynamic involvement of proteins in various processes, including cell proliferation, differentiation, and apoptosis. Here, we monitored the interactions between protein kinase-Cζ (PKCζ) and Bcl10 protein in untreated and etoposide (VP-16)-treated C4-I cells by means of a new combined morphological approach and validated it by taking stock of our previous proteomic and biochemical work (Chiarini et al. in J Proteome Res 11:3996-4012, 2012). We first analyzed the colocalization of PKCζ and Bcl10 proteins through classical immunofluorescent colocalization analysis. On the basis of these results, we developed a novel imaging approach combining immunofluorescence (IF) techniques with in situ PLA to identify the PKCζ·Bcl10 complexes at the level of a specific subcellular compartment, i.e., the nuclear envelope (NE). By this means, we could show that the amount of PKCζ·Bcl10 complexes localized at the NE of C4-I cells during proliferation or after treatment with VP-16 closely corresponded to our previous purely biochemical results. Hence, the present findings demonstrate that the combination of in situ PLA with classical IF detection is a novel powerful analytical tool allowing to morphologically demonstrate new specific protein-protein interactions at level of subcellular organelles, the complexes functions of which can next be clarified through proteomic/biochemical approaches.
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Técnica del Anticuerpo Fluorescente , Mapeo de Interacción de Proteínas/métodos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Células Cultivadas , Humanos , Rayos Láser , Microscopía Confocal , Unión Proteica , Proteína Quinasa C/metabolismoRESUMEN
Objectives: The optimal healing of skin wounds, deep burns, and chronic ulcers is an important clinical problem. Attempts to solve it have been driving the search for skin equivalents based on synthetic or natural polymers. Methods: Consistent with this endeavor, we used regenerated silk fibroin (SF) from Bombyx mori to produce a novel compound scaffold by welding a 3D carded/hydroentangled SF-microfiber-based nonwoven layer (C/H-3D-SFnw; to support dermis engineering) to an electrospun 2D SF nanofiber layer (ESFN; a basal lamina surrogate). Next, we assessed-via scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, mono- and co-cultures of HaCaT keratinocytes and adult human dermal fibroblasts (HDFs), dsDNA assays, exosome isolation, double-antibody arrays, and angiogenesis assays-whether the C/H-3D-SFnws/ESFNs would allow the reconstitution of a functional human skin analog in vitro. Results: Physical analyses proved that the C/H-3D-SFnws/ESFNs met the requirements for human soft-tissue-like implants. dsDNA assays revealed that co-cultures of HaCaTs (on the 2D ESFN surface) and HDFs (inside the 3D C/H-3D-SFnws) grew more intensely than did the respective monocultures. Double-antibody arrays showed that the CD9+/CD81+ exosomes isolated from the 14-day pooled growth media of HDF and/or HaCaT mono- or co-cultures conveyed 35 distinct angiogenic/growth factors (AGFs). However, versus monocultures' exosomes, HaCaT/HDF co-cultures' exosomes (i) transported larger amounts of 15 AGFs, i.e., PIGF, ANGPT-1, bFGF, Tie-2, Angiogenin, VEGF-A, VEGF-D, TIMP-1/-2, GRO-α/-ß/-γ, IL-1ß, IL-6, IL-8, MMP-9, and MCP-1, and (ii) significantly more strongly stimulated human dermal microvascular endothelial cells to migrate and assemble tubes/nodes in vitro. Conclusions: Our results showed that both cell-cell and cell-SF interactions boosted the exosomal release of AGFs from HaCaTs/HDFs co-cultured on C/H-3D-SFnws/ESFNs. Hence, such exosomes are an asset for prospective clinical applications as they advance cell growth and neoangiogenesis and consequently graft take and skin healing. Moreover, this new integument analog could be instrumental in preclinical and translational studies on human skin pathophysiology and regeneration.
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Fibroínas , Femenino , Humanos , Fibroínas/farmacología , Fibroínas/química , Técnicas de Cocultivo , Andamios del Tejido/química , Ingeniería de Tejidos/métodos , Células Endoteliales , Estudios Prospectivos , Factor de Crecimiento Placentario/metabolismo , Queratinocitos/fisiología , Fibroblastos/metabolismoRESUMEN
Increasingly prevalent acute and chronic human brain diseases are scourges for the elderly. Besides the lack of therapies, these ailments share a neuroinflammation that is triggered/sustained by different innate immunity-related protein oligomers called inflammasomes. Relevant neuroinflammation players such as microglia/monocytes typically exhibit a strong NLRP3 inflammasome activation. Hence the idea that NLRP3 suppression might solve neurodegenerative ailments. Here we review the recent Literature about this topic. First, we update conditions and mechanisms, including RNAs, extracellular vesicles/exosomes, endogenous compounds, and ethnic/pharmacological agents/extracts regulating NLRP3 function. Second, we pinpoint NLRP3-activating mechanisms and known NLRP3 inhibition effects in acute (ischemia, stroke, hemorrhage), chronic (Alzheimer's disease, Parkinson's disease, Huntington's disease, MS, ALS), and virus-induced (Zika, SARS-CoV-2, and others) human brain diseases. The available data show that (i) disease-specific divergent mechanisms activate the (mainly animal) brains NLRP3; (ii) no evidence proves that NLRP3 inhibition modifies human brain diseases (yet ad hoc trials are ongoing); and (iii) no findings exclude that concurrently activated other-than-NLRP3 inflammasomes might functionally replace the inhibited NLRP3. Finally, we highlight that among the causes of the persistent lack of therapies are the species difference problem in disease models and a preference for symptomatic over etiologic therapeutic approaches. Therefore, we posit that human neural cell-based disease models could drive etiological, pathogenetic, and therapeutic advances, including NLRP3's and other inflammasomes' regulation, while minimizing failure risks in candidate drug trials.
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Just as biomarkers specific for diseases, biomarkers indicative of healthy conditions are valuable for the early diagnosis, monitoring, and prognosis of diseases. Our study focused on discovering via proteomics a stable panel of urinary proteins in the human healthy population. Urine samples were collected three times during 4 months from 100 male and 100 female healthy donors and analyzed through four different fractionation techniques (i.e. in-gel, 2D-LC, OFFGEL, and mRP) coupled with HPLC-Chip-MS/MS. Thus, 1641 urinary proteins were identified with a high confidence, among which 70 exhibiting an intergender/day variation <0.25 were selected and matched with the previously published five largest urinary proteomes to get 56 candidate proteins. Next, a panel comprising 18 intact urinary proteins was constructed by comparing the urinary proteomes via SDS-PAGE and 2DE. Finally, such 18 urinary proteins were validated via enzyme-linked immunosorbent assay in eight healthy individuals. Most of these proteins had been related to multiple rather than to single diseases. Therefore, we surmise that this protein set could be used as a biomarker to assess the human health status. Further determinations of the normal fluctuations of the single urinary proteins in this series using samples from large numbers of healthy individuals are required prior to any application in clinical settings.
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Proteinuria/orina , Proteoma/análisis , Adolescente , Análisis de Varianza , Biomarcadores/orina , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Espectrometría de Masas , Proteómica/métodos , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Many features of deadly human cervical cancers (HCCs) still require elucidation. Among HCC-derived cell lines, here we used the C4-I one since its quantitative gene expression pattern most closely mimics invasive HCCs, including protein kinase-Cζ (PKCζ) overexpression. Via proteomic, bioinformatic, and biochemical approaches we identified 31 and 33 proteins co-immunoprecipitating with PKCζ from nuclear membranes (NMs) of, respectively, untreated or VP-16-exposed C4-I cells. Such proteins belonged to eight functional groups, whose compositions and relative sizes changed with either context. Of the 56 proteins identified, only eight were shared between the two subproteomes, including Bcl10. Surprisingly, proteins known to associate with Bcl10, like Carma1/3 and Malt1, in so-called CBM signalosomes were absent. Notably, in VP-16-treated C4-I cells, PKCζâ¢Bcl10 complexes increasingly accrued at NMs, where PKCζ phosphorylated Bcl10, as PKCζ also did in vitro and in cell-free systems, both processes being thwarted by interfering RNA (iRNA) PKCζ depletion. Caspase-3 was associated with PKCζâ¢Bcl10 complexes and proteolyzed PKCζ leading to its inactivation/destruction; both events were prevented by Bcl10 iRNA suppression. Thus, PKCζ's molecular interactions and functional roles changed strikingly according to the untreated or apoptogen-treated cells context, and by complexing with Bcl10, PKCζ surprisingly favored its own demise, which suggests both proteins as HCCs therapeutic targets.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Membrana Nuclear/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos Fitogénicos/farmacología , Proteína 10 de la LLC-Linfoma de Células B , Caspasa 3/metabolismo , Línea Celular Tumoral , Etopósido/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunoprecipitación , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Proteína Quinasa C/genética , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Proteómica , Interferencia de ARN , Neoplasias del Cuello UterinoRESUMEN
The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatin receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75(NTR)). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both Aß(1-42) and the mutant human tau protein tau(P301L,) the dentate granule cells still had immunostainable SSTR3- and p75(NTR)-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of Aß(1-42) or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of Aß(1-42) and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Giro Dentado/metabolismo , Fragmentos de Péptidos/biosíntesis , Proteínas tau/biosíntesis , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Animales , Biomarcadores/metabolismo , Cilios/metabolismo , Giro Dentado/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores de Somatostatina/metabolismo , Coloración y Etiquetado , Proteínas tau/genéticaRESUMEN
Human neuroinflammatory and neurodegenerative diseases, whose prevalence keeps rising, are still unsolved pathobiological/therapeutical problems. Among others, recent etiology hypotheses stressed as their main driver a chronic neuroinflammation, which is mediated by innate immunity-related protein oligomers: the inflammasomes. A panoply of exogenous and/or endogenous harmful agents activates inflammasomes' assembly, signaling, and IL-1ß/IL-18 production and neural cells' pyroptotic death. The underlying concept is that inflammasomes' chronic activation advances neurodegeneration while their short-lasting operation restores tissue homeostasis. Hence, from a therapeutic standpoint, it is crucial to understand inflammasomes' regulatory mechanisms. About this, a deluge of recent studies focused on the NLRP3 inflammasome with suggestions that its pharmacologic block would hinder neurodegeneration. Yet hitherto no evidence proves this view. Moreover, known inflammasomes are numerous, and the mechanisms regulating their expression and function may vary with the involved animal species and strains, as well as organs and cells, and the harmful factors triggered as a result. Therefore, while presently leaving out some little-studied inflammasomes, this review focuses on the "other than NLRP3" inflammasomes that participate in neuroinflammation's complex mechanisms: NLRP1, NLRP2, NLRC4, and AIM2. Although human-specific data about them are relatively scant, we stress that only a holistic view including several human brain inflammasomes and other potential pathogenetic drivers will lead to successful therapies for neuroinflammatory and neurodegenerative diseases.
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BACKGROUND: Our earlier works showed the quick vascularization of mouse skin grafted Bombyx mori 3D silk fibroin nonwoven scaffolds (3D-SFnws) and the release of exosomes enriched in angiogenic/growth factors (AGFs) from in vitro 3D-SFnws-stuck human dermal fibroblasts (HDFs). Here, we explored whether coronary artery adult human smooth muscle cells (AHSMCs) also release AGFs-enriched exosomes when cultured on 3D-SFnws in vitro. METHODS: Media with exosome-depleted FBS served for AHSMCs and human endothelial cells (HECs) cultures on 3D-SFnws or polystyrene. Biochemical methods and double-antibody arrays assessed cell growth, metabolism, and intracellular TGF-ß and NF-κB signalling pathways activation. AGFs conveyed by CD9+/CD81+ exosomes released from AHSMCs were double-antibody array analysed and their angiogenic power evaluated on HECs in vitro. RESULTS: AHSMCs grew and consumed D-glucose more intensely and showed a stronger phosphorylation/activation of TAK-1, SMAD-1/-2/-4/-5, ATF-2, c-JUN, ATM, CREB, and an IκBα phosphorylation/inactivation on SFnws vs. polystyrene, consistent overall with a proliferative/secretory phenotype. SFnws-stuck AHSMCs also released exosomes richer in IL-1α/-2/-4/-6/-8; bFGF; GM-CSF; and GRO-α/-ß/-γ, which strongly stimulated HECs' growth, migration, and tubes/nodes assembly in vitro. CONCLUSIONS: Altogether, the intensified AGFs exosomal release from 3D-SFnws-attached AHSMCs and HDFs could advance grafts' colonization, vascularization, and take in vivo-noteworthy assets for prospective clinical applications.
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Background: Skin cutaneous melanoma (SKCM) is the deadliest skin cancer and has the most rapidly increasing incidences among all cancer types. Previous research elucidated that melanoma can only be successfully treated with surgical abscission in the early stage. Therefore, reliable and specific biomarkers are crucial to melanoma diagnosis since it often looks like nevi in the clinical manifestations. Moreover, identifying key genes contributing to melanoma progression is also highly regarded as a potential strategy for melanoma therapy. In this respect, translation initiator eIF6 has been proved as a pro-tumor factor in several cancers. However, the role of eIF6 in the skin cutaneous melanoma progression and its potential as a prognostic marker is still unexplored. Methods: The immunochemical analysis of clinical specimens were served to assess eIF6 expression levels. Gene Expression Profiling Interactive Analysis (GEPIA) database consultations allowed us to find the survival rates of the eIF6-overexpressed patients. eIF6 cellular effects were evaluated in an eIF6-overexpressed A375 cell line constructed with a lentivirus. The analysis of down-stream effectors or pathways was conducted using C-Bioportal and STRING databases. Results: Our results revealed that eIF6 was highly over-expressed in melanomas compared to normal skin specimens, and thus the abnormally high level of eIF6 can be a diagnostic marker for melanoma. The in silica analysis indicated that patients with eIF6 over-expression had lower survival rates than that low-expression in SKCM. Meanwhile, similar results also could be found in the other four types of cancers. In vitro, over-expression of eIF6 increased the proliferation and migration of melanoma cells. Correspondingly, pan-cancer clustering analysis indicated the expression level of intermediate filament proteins was correlated with that of eIF6 expression. In our study, all over-expressed keratin proteins, in accordance with over-expressed eIF6, had a negative correlation with melanoma prognosis. Moreover, the decreased methylation level of keratin genes suggested a new potential regulation mode of eIF6. Conclusions: The up-regulated eIF6 could be a potential diagnostic and prognostic biomarker of melanoma. This study also provides insights into the potential role of eIF6 in pan-cancer epigenetic regulation.
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BACKGROUND: Rapid diagnosis of microbes in the burn wound is a big challenge in the medical field. Traditional biochemical detection techniques take hours or days to identify the species of contaminating and drug-resistant microbes. Near-infrared spectroscopy (NIRS) is evaluated to address the need for a fast and sensitive method for the detection of bacterial contamination in liquids. METHODS: Herin, we developed a novel technique which by using NIRS together with supporting vector machine (SVM), to identify the microbial species and drug-resistant microbes in LB medium, and to diagnose the wound colonization and wound infection models of pigs. RESULTS: The device could recognize 100% of seven kinds of microbes and 99.47% of the multi-drug resistant Staphylococcus aureus (S. aureus), with a concentration of 109 cfu ml-1 in LB medium. The accuracy of the microbial identification in colonized and infected wounds in-situ was 100%. The detection limit of NIRS with SVM for the detection of S. aureus and Escherichia coli (E. coli) was 101 cfu ml-1 in LB medium. Identification time was less than 5 s. CONCLUSION: Our findings validate for the first time a novel technique aimed at the rapid, noncontacted, highly sensitive, and specific recognition of several microbial species including drug-resistant ones. This technique could represent a promising approach to identify diverse microbial species and a potential bedside device to rapidly diagnose infected wounds.
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Quemaduras , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Infección de Heridas , Animales , Quemaduras/microbiología , Escherichia coli , Humanos , Espectroscopía Infrarroja Corta , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus , Porcinos , Infección de Heridas/diagnóstico , Infección de Heridas/microbiologíaRESUMEN
BACKGROUND: Bombyx mori silk fibroin is a biomacromolecule that allows the assembly of scaffolds for tissue engineering and regeneration purposes due to its cellular adhesiveness, high biocompatibility and low immunogenicity. Earlier work showed that two types of 3D silk fibroin nonwovens (3D-SFnws) implanted into mouse subcutaneous tissue were promptly vascularized via undefined molecular mechanisms. The present study used nontumorigenic adult human dermal fibroblasts (HDFs) adhering to a third type of 3D-SFnws to assess whether HDFs release exosomes whose contents promote neoangiogenesis. METHODS: Electron microscopy imaging and physical tests defined the features of the novel carded/hydroentangled 3D-SFnws. HDFs were cultured on 3D-SFnws and polystyrene plates in an exosome-depleted medium. DNA amounts and D-glucose consumption revealed the growth and metabolic activities of HDFs on 3D-SFnws. CD9-expressing total exosome fractions were from conditioned media of 3D-SFnws and 2D polystyrene plates HDF cultures. Angiogenic growth factors (AGFs) in equal amounts of the two groups of exosomal proteins were analysed via double-antibody arrays. A tube formation assay using human dermal microvascular endothelial cells (HDMVECs) was used to evaluate the exosomes' angiogenic power. RESULTS: The novel features of the 3D-SFnws met the biomechanical requirements typical of human soft tissues. By experimental day 15, 3D-SFnws-adhering HDFs had increased 4.5-fold in numbers and metabolized 5.4-fold more D-glucose than at day 3 in vitro. Compared to polystyrene-stuck HDFs, exosomes from 3D-SFnws-adhering HDFs carried significantly higher amounts of AGFs, such as interleukin (IL)-1α, IL-4 and IL-8; angiopoietin-1 and angiopoietin-2; angiopoietin-1 receptor (or Tie-2); growth-regulated oncogene (GRO)-α, GRO-ß and GRO-γ; matrix metalloproteinase-1; tissue inhibitor metalloproteinase-1; and urokinase-type plasminogen activator surface receptor, but lesser amounts of anti-angiogenic tissue inhibitor metalloproteinase-2 and pro-inflammatory monocyte chemoattractant protein-1. At concentrations from 0.62 to 10 µg/ml, the exosomes from 3D-SFnws-cultured HDFs proved their angiogenic power by inducing HDMVECs to form significant amounts of tubes in vitro. CONCLUSIONS: The structural and mechanical properties of carded/hydroentangled 3D-SFnws proved their suitability for tissue engineering and regeneration applications. Consistent with our hypothesis, 3D-SFnws-adhering HDFs released exosomes carrying several AGFs that induced HDMVECs to promptly assemble vascular tubes in vitro. Hence, we posit that once implanted in vivo, the 3D-SFnws/HDFs interactions could promote the vascularization and repair of extended skin wounds due to burns or other noxious agents in human and veterinary clinical settings.
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The accurate and objective evaluation of burn depth is a significant challenge in burn wound care. Herein, we used near infrared spectroscopy (NIRS) technology to measure the different depth of thermal burns in ex vivo porcine models. Based on the intensity of the spectral signals and the diffuse reflection theory, we extracted the optical parameters involved in functional (total hemoglobin and water content) and structural (tissue scattered size and scattered particles) features that reflect the changes in burn depth. Next, we applied support vector regression to construct a model including the optical property parameters and the burn depth. Finally, we histologically verified the burn depth data collected via NIRS. The results showed that our inversion model could achieve an average relative error of about 7.63%, while the NIRS technology diagnostic accuracy was in the range of 50 µm. For the first time, this novel technique provides physicians with real-time burn depth information objectively and accurately.
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The neurodegenerative changes in Alzheimer's disease (AD) are elicited by the accumulation of beta-amyloid peptides (Abeta), which damage neurons either directly by interacting with components of the cell surface to trigger cell death signaling or indirectly by activating astrocytes and microglia to produce inflammatory mediators. It has been recently proposed that the p75 neurotrophin receptor (p75(NTR)) is responsible for neuronal damage by interacting with Abeta. By using neuroblastoma cell clones lacking the expression of all neurotrophin receptors or engineered to express full-length or various truncated forms of p75(NTR), we could show that p75(NTR) is involved in the direct signaling of cell death by Abeta via the function of its death domain. This signaling leads to the activation of caspases-8 and -3, the production of reactive oxygen intermediates and the induction of an oxidative stress. We also found that the direct and indirect (inflammatory) mechanisms of neuronal damage by Abeta could act synergistically. In fact, TNF-alpha and IL-1beta, cytokines produced by Abeta-activated microglia, could potentiate the neurotoxic action of Abeta mediated by p75(NTR) signaling. Together, our results indicate that neurons expressing p75(NTR), mostly if expressing also proinflammatory cytokine receptors, might be preferential targets of the cytotoxic action of Abeta in AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Supervivencia Celular/efectos de los fármacos , Citocinas/toxicidad , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Receptores de Factor de Crecimiento Nervioso/fisiología , Células Clonales , Sinergismo Farmacológico , Regulación de la Expresión Génica , Humanos , Cinética , Neuroblastoma , Neuronas/patología , Fragmentos de Péptidos/toxicidad , Receptor de Factor de Crecimiento Nervioso , Receptor trkA/genética , Receptor trkA/fisiología , Receptores de Factor de Crecimiento Nervioso/efectos de los fármacos , Receptores de Factor de Crecimiento Nervioso/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transfección , Células Tumorales CultivadasRESUMEN
Chronic wounds caused by infections, diabetes, and radiation exposures are becoming a worldwide growing medical burden. Recent progress highlighted the physical signals determining stem cell fates and bacterial resistance, which holds potential to achieve a better wound regeneration in situ. Nanoparticles (NPs) would benefit chronic wound healing. However, the cytotoxicity of the silver NPs (AgNPs) has aroused many concerns. This review targets the tunable physical properties (i.e., mechanical-, structural-, and size-related properties) of either dermal matrixes or wound dressings for chronic wound care. Firstly, we discuss the recent discoveries about the mechanical- and structural-related regulation of stem cells. Specially, we point out the currently undocumented influence of tunable mechanical and structural properties on either the fate of each cell type or the whole wound healing process. Secondly, we highlight novel dermal matrixes based on either natural tropoelastin or synthetic elastin-like recombinamers (ELRs) for providing elastic recoil and resilience to the wounded dermis. Thirdly, we discuss the application of wound dressings in terms of size-related properties (i.e., metal NPs, lipid NPs, polymeric NPs). Moreover, we highlight the cytotoxicity of AgNPs and propose the size-, dose-, and time-dependent solutions for reducing their cytotoxicity in wound care. This review will hopefully inspire the advanced design strategies of either dermal matrixes or wound dressings and their potential therapeutic benefits for chronic wounds.