Engineering nano-drug biointerface to overcome biological barriers toward precision drug delivery.

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.

Danger-Sensing/Patten Recognition Receptors and Neuroinflammation in Alzheimer's Disease.

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.

Evidence for caspase-dependent programmed cell death along with repair processes in affected skeletal muscle fibres in patients with mitochondrial disorders.

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.

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aß42 prompted by exogenous fibrillary or soluble Aß25-35 in human cortical astrocytes and neurons-therapeutic relevance to Alzheimer's disease.

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.

Combining immunofluorescence with in situ proximity ligation assay: a novel imaging approach to monitor protein-protein interactions in relation to subcellular localization.

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.

Human Keratinocytes and Fibroblasts Co-Cultured on Silk Fibroin Scaffolds Exosomally Overrelease Angiogenic and Growth Factors.

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.

NLRP3 Inflammasome's Activation in Acute and Chronic Brain Diseases-An Update on Pathogenetic Mechanisms and Therapeutic Perspectives with Respect to Other Inflammasomes.

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.

Role-shifting PKCζ fosters its own proapoptotic destruction by complexing with Bcl10 at the nuclear envelope of human cervical carcinoma cells: a proteomic and biochemical study.

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, PKCζ•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 PKCζ•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.

"Other Than NLRP3" Inflammasomes: Multiple Roles in Brain Disease.

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.

Adult Human Vascular Smooth Muscle Cells on 3D Silk Fibroin Nonwovens Release Exosomes Enriched in Angiogenic and Growth-Promoting Factors.

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.

Electrospun Silk Fibroin Scaffolds for Tissue Regeneration: Chemical, Structural, and Toxicological Implications of the Formic Acid-Silk Fibroin Interaction.

The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm-1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 µmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 µmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

Exosomes of adult human fibroblasts cultured on 3D silk fibroin nonwovens intensely stimulate neoangiogenesis.

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.

The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells.

The densely ciliated granule cell layer of the adult murine hippocampal dentate gyrus is one of two sites of adult neurogenesis. The granule cells have already been proven to localize their SSTR3 (somatostatin receptor 3) receptors to their so-called primary cilia. Here we show for the first time that 70-90% of these cells in 7-18 months-old wild-type and 3×Tg-AD (Alzheimer disease transgenic) mice also load p75(NTR) receptors into the structures containing SSTR3, i.e., their primary cilia. On the other hand, p75(NTR')s TrkA co-receptors were not localized to cilia but conventionally distributed throughout the cell surface. Significantly fewer cells (20-40%) in the hippocampal CA1 and CA3 regions and cerebral cortex have p75(NTR) containing cilia. While we don't know what the impact of the cilial localization of p75(NTR) on dentate gyral adult neurogenesis and memory encoding might be, the cilia's amyloid ß-activatable p75(NTR) receptors could be damaging or lethal to the hippocampal functioning of amyloid ß-accumulating Alzheimer brain.

CaSR Antagonist (Calcilytic) NPS 2143 Hinders the Release of Neuroinflammatory IL-6, Soluble ICAM-1, RANTES, and MCP-2 from Aß-Exposed Human Cortical Astrocytes.

Available evidence shows that human cortical neurons' and astrocytes' calcium-sensing receptors (CaSRs) bind Amyloid-beta (Aß) oligomers triggering the overproduction/oversecretion of several Alzheimer's disease (AD) neurotoxinseffects calcilytics suppress. We asked whether AßCaSR signaling might also play a direct pro-neuroinflammatory role in AD. Cortical nontumorigenic adult human astrocytes (NAHAs) in vitro were untreated (controls) or treated with Aß25-35±ï€ NPS 2143 (a calcilytic) and any proinflammatory agent in their protein lysates and growth media assayed via antibody arrays, enzyme-linked immunosorbent assays (ELISAs), and immunoblots. Results show Aß•CaSR signaling upregulated the synthesis and release/shedding of proinflammatory interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1) (holoprotein and soluble [s] fragment), Regulated upon Activation, normal T cell Expressed and presumably Secreted (RANTES), and monocyte chemotactic protein (MCP)-2. Adding NPS 2143 (i) totally suppressed IL-6's oversecretion while remarkably reducing the other agents' over-release; and (ii) more effectively than Aß alone increased over controls the four agents' distinctive intracellular accumulation. Conversely, NPS 2143 did not alter Aß-induced surges in IL-1ß, IL-3, IL-8, and IL-16 secretion, consequently revealing their Aß•CaSR signaling-independence. Finally, Aß25-35±ï€ NPS 2143 treatments left unchanged MCP-1's and TIMP-2's basal expression. Thus, NAHAs Aß•CaSR signaling drove four proinflammatory agents' over-release that NPS 2143 curtailed. Therefore, calcilytics would also abate NAHAs' Aß•CaSR signaling direct impact on AD's neuroinflammation.

Maladaptive remodeling of pulmonary artery root autografts after Ross procedure: A proteomic study.

OBJECTIVE: Pulmonary autograft root dilatation is the major long-term complication after Ross procedure and the leading cause for reoperation. However, the mechanisms underlying dilatation remain to be elucidated. This study analyzed the proteomic changes seen in the dilated pulmonary autograft compared with normal pulmonary artery and aorta tissues. METHODS: Pulmonary autograft surgical samples were taken from 9 consecutive patients (mean age 37 ± 14; 15-51 years) with mean diameters of 5.2 ± 0.5 cm (4.6-5.8 cm) reoperated 8 to 16 years after Ross procedure. Control pulmonary artery and aorta samples were from 7 age- and sex-matched cardiac donors. Tunicae mediae from all samples were processed for proteomic analysis via 2-dimensional electrophoresis, matrix-assisted-laser-desorption-ionization-time of flight/mass spectrometry, and bioinformatics. The thus-identified putatively relevant proteins were validated via Western immunoblotting. RESULTS: Pulmonary autograft proteome features differed markedly from control pulmonary arteries, since proteins related to focal adhesions (eg, paxillin), cytoskeleton (eg, vimentin), and metalloprotease-regulating proteoglycans (eg, testican-2) were significantly up-regulated, whereas significant decreases occurred in microfibril-associated glycoprotein1, which controls elastic fiber buildup. Profound changes also occurred in cell-signaling proteins, ie, increases in soluble Jagged-1 fragment and ectodysplasin-2 receptor, and decreases in Notch-1 intracellular domain fragment. Moreover, pulmonary autograft expression levels of Paxillin, Vimentin, Jagged-1 fragment, and Notch1 intracellular domain fragment also differed from those of control aorta. CONCLUSIONS: This study provides the first description of the specific proteomic features of dilated pulmonary autograft tunica media, which separate them sharply not only from those of control pulmonary artery and aorta but also of aortic aneurysms. These findings suggest that dilated pulmonary autografts undergo a unique maladaptive remodeling process deserving further investigation.

Corrigendum to: 'Mesenchymal stromal cells-exosomes: a promising cell-free therapeutic tool for wound healing and cutaneous regeneration'.

[This corrects the article DOI: 10.1186/s41038-019-0178-8.].

Proteomic analysis of GTP cyclohydrolase 1 multiprotein complexes in cultured normal adult human astrocytes under both basal and cytokine-activated conditions.

GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme of a metabolic pathway synthesizing tetrahydrobiopterin (BH(4)), the cofactor dimerizing and activating inducible nitric oxide synthase (NOS-2). GCH1 protein expression and enzyme activity are minimal in cultured, phenotypically stable, untreated normal adult human astrocytes (NAHA), but are strongly induced, together with NOS-2, by a mixture of three proinflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma--the CM-trio) released by microglia under brain-damaging conditions. The resulting hyper-production of NO severely harms neurons. In this study, using MALDI-TOF/MS, PMF, Western immunoblotting (WB), and antibody microarrays we identified several proteins coimmunoprecipitating with GCH1. Under basal conditions, GCH1 was associated with various adaptor/regulator molecules involved in G-protein-coupled receptors signalling, protein serine/threonine phosphatase 2Cbeta (PP2Cbeta), and serine-threonine kinases like Ca(2+) calmodulin kinases (CaMKs), casein kinases (CKs), cAMP-dependent kinases (PKAs), and mitogen-activated protein kinases (MAPKs). Exposure to the three cytokines' mixture (CM-trio) significantly changed, within the 48-72 h required for the induction and activation of GCH1, the levels and identities of some of the 0 h-associated proteins: after 72 h CK-IIalpha tended to dissociate from, whereas MAPK12 and JNK3 were strongly associated with fully active GCH1. These findings provide a first enticing glimpse into the intricate mechanisms regulating GCH1 activation by proinflammatory cytokines in NAHA, and may have therapeutic implications.

Photoexcited calphostin C selectively destroys nuclear lamin B1 in neoplastic human and rat cells - a novel mechanism of action of a photodynamic tumor therapy agent.

Lamin B1, a major component of the nuclear lamina, anchors the nucleus to the cytoskeletal cage, and controls nuclear orientation, chromosome positioning and, alongside several enzymes, fundamental nuclear functions. Exposing polyomavirus-transformed rat pyF111 fibroblasts and human cervical carcinoma (HCC) C4-I cells for 30 min to photoexcited perylenequinone calphostin C, i.e. Cal C(phiE), an established reactive oxygen species (ROS)-generator and protein kinase C (PKC) inhibitor, caused the cells to selectively oxidize and then totally destroy their nuclear lamin B1 by only 60 min after starting the treatment, i.e. when apoptotic caspases' activities had not yet increased. However, while the oxidized lamin B1 was being destroyed, lamins A/C, the lamin A-associated nuclear envelope protein emerin, and the nucleoplasmic protein cyclin E were neither oxidized nor destroyed. The oxidized lamin B was ubiquitinated and demolished in the proteasome probably by an enhanced peptidyl-glutaminase-like activity. Hence, the Cal C(phiE)-induced rapid and selective lamin B1 oxidation and proteasomal destruction ahead of the activation of apoptotic caspases was by itself a most severe molecular lesion impairing vital nuclear functions. Conversely, Cal C directly added to the cells kept in the dark damaged neither nuclear lamin B1 nor cell viability. Thus, our findings reveal a novel cell-damaging mechanism of a photodynamic tumor therapeutic agent.

Family C G-Protein-Coupled Receptors in Alzheimer's Disease and Therapeutic Implications.

Alzheimer's disease (AD), particularly its sporadic or late-onset form (SAD/LOAD), is the most prevalent (96-98% of cases) neurodegenerative dementia in aged people. AD's neuropathology hallmarks are intrabrain accumulation of amyloid-ß peptides (Aßs) and of hyperphosphorylated Tau (p-Tau) proteins, diffuse neuroinflammation, and progressive death of neurons and oligodendrocytes. Mounting evidences suggest that family C G-protein-coupled receptors (GPCRs), which include γ-aminobutyric acid B receptors (GABABRs), metabotropic glutamate receptors (mGluR1-8), and the calcium-sensing receptor (CaSR), are involved in many neurotransmitter systems that dysfunction in AD. This review updates the available knowledge about the roles of GPCRs, particularly but not exclusively those expressed by brain astrocytes, in SAD/LOAD onset and progression, taking stock of their respective mechanisms of action and of their potential as anti-AD therapeutic targets. In particular, GABABRs prevent Aßs synthesis and neuronal hyperexcitability and group I mGluRs play important pathogenetic roles in transgenic AD-model animals. Moreover, the specific binding of Aßs to the CaSRs of human cortical astrocytes and neurons cultured in vitro engenders a pathological signaling that crucially promotes the surplus synthesis and release of Aßs and hyperphosphorylated Tau proteins, and also of nitric oxide, vascular endothelial growth factor-A, and proinflammatory agents. Concurrently, Aßs•CaSR signaling hinders the release of soluble (s)APP-α peptide, a neurotrophic agent and GABABR1a agonist. Altogether these effects progressively kill human cortical neurons in vitro and likely also in vivo. Several CaSR's negative allosteric modulators suppress all the noxious effects elicited by Aßs•CaSR signaling in human cortical astrocytes and neurons thus safeguarding neurons' viability in vitro and raising hopes about their potential therapeutic benefits in AD patients. Further basic and clinical investigations on these hot topics are needed taking always heed that activation of the several brain family C GPCRs may elicit divergent upshots according to the models studied.

Over Expressed TKTL1, CIP-2A, and B-MYB Proteins in Uterine Cervix Epithelium Scrapings as Potential Risk Predictive Biomarkers in HR-HPV-Infected LSIL/ASCUS Patients.

High oncogenic risk human papillomaviruses (HR-HPVs) promote cervical carcinoma development, the fourth most common feminine cancer. A slow oncodevelopmental phase-defined histopathologically as Cervical Intraepithelial Neoplasia (CIN) grades 1-3, or cytologically as Low- or High-grade Squamous Intraepithelial Lesions (LSIL or HSIL)-precedes the malignancy. Cervical carcinoma screenings through HR-HPV genotyping and Pap smears are regularly performed in Western countries. Faulty cytology screening or genotyping or patients' non-compliance with follow-ups can let slip an oncoprogression diagnosis. Novel biomarker tests flanking HR-HPV genotyping and cytology could objectively predict the risk of disease progression thus helping triage LSIL/ASCUS patients. Here, anonymized leftovers of fresh cervical epithelium scrapings from twice (LSIL/ASCUS and HR-HPV DNA)-positive and twice (Pap smear- and HR-HPV DNA)-negative (control) patients in a proteome-preserving solution served to assess the biomarker worth of three cervical carcinoma-related proteins, i.e., B-MYB (or MYBL2), Cancerous Inhibitor of PP2A (CIP-2a), and transketolase-like1 (TKTL1). Leftovers anonymity was strictly kept and storage at -80°C, protein extraction, immunoblotting, and band densitometry were blindly performed. Only after tests completion, the anonymous yet code-corresponding HR-HPV-genotyping and cytology data allowed to assign each sample to the twice-positive or twice-negative group. Descriptive statistics showed that the three proteins levels significantly increased in the twice-positive vs. twice-negative scrapings. Diagnostic ROC curve analysis identified each protein's Optimal Decision Threshold (OTD) showing that TKTL1 and CIP-2a are stronger risk predictive biomarkers (Sensitivity, 0.91-0.93; Specificity, 0.77-0.83) than B-MYB. Logistic Regression coupled with Likelihood-Ratio Tests confirmed that a highly significant relation links increasing TKTL1/CIP-2a/B-MYB protein levels in twice-positive cervical scrapings to the risk of HR-HPV-driven oncoprogression. Finally, a 3 year clinical follow-up showed that 13 patients (50% of total) of the twice-positive group with biomarker values over OTDs compliantly underwent scheduled colposcopy and biopsy. Of these, 11 (i.e., 84.7%) received a positive histological diagnosis, i.e., CIN1 (n = 5; 38.5%) or CIN2/CIN2+ (n = 6; 46,2%). Therefore, TKTL1/CIP-2a/B-MYB protein levels could objectively predict oncoprogression risk in twice (HR-HPV- and Pap smear)-positive women. Further studies will assess the translatability of these findings into clinical settings.