3D-printed biosensors in biomedical applications exploiting plasmonic phenomena and antibody self-assembled monolayers.

In this work, a 3D-printed plasmonic chip based on a silver-gold bilayer was developed in order to enhance the optical response of the surface plasmon resonance (SPR) probe. More specifically, numerical and experimental results were obtained on the 3D-printed SPR platform based on a silver-gold bilayer. Then, the optimized probe's gold plasmonic interface was functionalized with a specific antibody directed against the p27Kip1 protein (p27), an important cell cycle regulator. The 3D-printed plasmonic biosensor was tested for p27 detection with good selectivity and a detection limit of 55 pM. The biosensor system demonstrated performance similar to commercially available ELISA (enzyme-linked immunoassay) kits, with several advantages, such as a wide detection range and a modular and simple-based architecture. The proposed biosensing technology offers flexible deployment options that are useful in disposable, low-cost, small-size, and simple-to-use biochips, envisaging future applications in experimental and biomedical research.

Plasmon resonance biosensor for interleukin-1ß point-of-care determination: A tool for early periodontitis diagnosis.

Among pro-inflammatory cytokines, Interleukin-1ß is crucially involved in several inflammatory-based diseases and even cancer. Increased Interleukin-1ß levels in oral fluids have been proposed as an early marker of periodontitis, a broadly diffused chronic inflammatory condition of periodontal-supporting tissues, leading eventually to tooth loss. We describe the development of a portable surface-plasmon-resonance-based optical fiber probe suitably coated with an anti-Interleukin-1ß antibody monolayer. A pico-nanomolar linear range of determination was obtained in both buffer solution and saliva with a rapid (3 min) incubation and high selectivity in presence of interferents. Higher Interleukin-1ß concentration in the saliva of a periodontitis patient compared to a healthy control was determined. These measurements were validated by an automated ELISA system. Our results sustain the potential applicability of the proposed SPR-POF as diagnostic point-of-care device for real-time monitoring of salivary Interleukin-1ß, that can support early detection of oral inflammatory-based pathologies and rapid and timely therapeutic decisions.

A novel plasmonic optical-fiber-based point-of-care test for periodontal MIP-1α detection.

The analysis of salivary biomarkers as expression of periodontal health conditions has been proposed as a useful aid to conventional diagnostic approaches. In this study, we present a point-of-care test (POCT) exploiting a surface plasmon resonance (SPR)-based optical biosensor to detect salivary macrophage inflammatory protein (MIP)-1α, a promising marker of periodontitis. A plastic optical fiber (POF) was suitably modified and functionalized by an antibody self-assembled monolayer against MIP-1α for plasmonic detection. The proposed SPR-POF biosensor showed high selectivity and very low limit of detection for MIP-1α of 129 fM (1.0 pg/mL) in phosphate-buffered saline and 346 fM (2.7 pg/mL) in saliva. As a proof of concept, this POCT was also able to discriminate between a periodontitis patient and a healthy subject. The obtained results support the future application of this technology for an on-site detection and real-time monitoring of periodontal health conditions for diagnostic and therapeutic purposes.

Genome editing and cancer therapy: handling the hypoxia-responsive pathway as a promising strategy.

The precise characterization of oxygen-sensing pathways and the identification of pO2-regulated gene expression are both issues of critical importance. The O2-sensing system plays crucial roles in almost all the pivotal human processes, including the stem cell specification, the growth and development of tissues (such as embryogenesis), the modulation of intermediate metabolism (including the shift of the glucose metabolism from oxidative to anaerobic ATP production and vice versa), and the control of blood pressure. The solid cancer microenvironment is characterized by low oxygen levels and by the consequent activation of the hypoxia response that, in turn, allows a complex adaptive response characterized mainly by neoangiogenesis and metabolic reprogramming. Recently, incredible advances in molecular genetic methodologies allowed the genome editing with high efficiency and, above all, the precise identification of target cells/tissues. These new possibilities and the knowledge of the mechanisms of adaptation to hypoxia suggest the effective development of new therapeutic approaches based on the manipulation, targeting, and exploitation of the oxygen-sensor system molecular mechanisms.

Hypocalcemia: A key biomarker in hospitalized COVID-19 patients.

BACKGROUND: At the end of 2019 a new respiratory syndrome emerged in China named Coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 infection. Considering the severity of the disease in adult subjects with one or more chronic pathologies, it was mandatory to find simple and effective biomarkers for negative prognosis of the disease easily available at the admission to the hospital. METHODS: To identify possible parameters showing association with the outcome in COVID-19 patients with pre-existing chronic diseases, blood biochemical profiles of 511 patients, enrolled from March to June 2020, were retrospectively evaluated. The pathological conditions taken into consideration were diabetes, arterial hypertension, chronic kidney disease, cardiovascular diseases, chronic obstructive pulmonary disease, obesity, and cancer. All the data were collected upon admission to the emergency room (ER) during the indicated period. RESULTS: We observed that serum and ionized calcium were prevalently altered in our cohort. We determined that hypocalcemia was a major parameter associated with mechanical ventilation and poor prognosis, correlating also with the presence of comorbidities such as cardiovascular diseases, chronic kidney disease, and cancer. In addition, we found a positive correlation between hypocalcemia and clinical complications during hospitalizations. CONCLUSIONS: Our results strengthen the relevance of serum calcium concentration as a useful prognostic biomarker in hospitalized COVID-19 patients.

An Unanticipated Modulation of Cyclin-Dependent Kinase Inhibitors: The Role of Long Non-Coding RNAs.

It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors' (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control.

p27Kip1, an Intrinsically Unstructured Protein with Scaffold Properties.

The Cyclin-dependent kinase (CDK) regulator p27Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes.

A Beckwith-Wiedemann-Associated CDKN1C Mutation Allows the Identification of a Novel Nuclear Localization Signal in Human p57Kip2.

p57Kip2 protein is a member of the CIP/Kip family, mainly localized in the nucleus where it exerts its Cyclin/CDKs inhibitory function. In addition, the protein plays key roles in embryogenesis, differentiation, and carcinogenesis depending on its cellular localization and interactors. Mutations of CDKN1C, the gene encoding human p57Kip2, result in the development of different genetic diseases, including Beckwith-Wiedemann, IMAGe and Silver-Russell syndromes. We investigated a specific Beckwith-Wiedemann associated CDKN1C change (c.946 C>T) that results in the substitution of the C-terminal amino acid (arginine 316) with a tryptophan (R316W-p57Kip2). We found a clear redistribution of R316W-p57Kip2, in that while the wild-type p57Kip2 mostly occurs in the nucleus, the mutant form is also distributed in the cytoplasm. Transfection of two expression constructs encoding the p57Kip2 N- and C-terminal domain, respectively, allows the mapping of the nuclear localization signal(s) (NLSs) between residues 220-316. Moreover, by removing the basic RKRLR sequence at the protein C-terminus (from 312 to 316 residue), p57Kip2 was confined in the cytosol, implying that this sequence is absolutely required for nuclear entry. In conclusion, we identified an unreported p57Kip2 NLS and suggest that its absence or mutation might be of relevance in CDKN1C-associated human diseases determining significant changes of p57Kip2 localization/regulatory roles.

A cancer-associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss-of-function.

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27Kip1 , a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer-associated germinal p27Kip1 variant, namely glycine9->arginine-p27Kip1 (G9R-p27Kip1 ) identified in a parathyroid adenoma. We unexpectedly found that G9R-p27Kip1 lacks the major tumor suppressor activities of p27Kip1 including its antiproliferative and pro-apoptotic functions. In addition, G9R-p27Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild-type p27Kip1 -transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R-p27Kip1 on S12, a residue normally never found modified in p27Kip1 . The novel S12 phosphorylation appears responsible for the loss of function of G9R-p27Kip1 since S12AG9R-p27Kip1 recovers most of the p27Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D-p27Kip1 recapitulates G9R-p27Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin-dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post-translational modifications due to generation of novel consensus sequences by cancer-associated missense mutations.

Regulation of p27Kip1 and p57Kip2 Functions by Natural Polyphenols.

In numerous instances, the fate of a single cell not only represents its peculiar outcome but also contributes to the overall status of an organism. In turn, the cell division cycle and its control strongly influence cell destiny, playing a critical role in targeting it towards a specific phenotype. Several factors participate in the control of growth, and among them, p27Kip1 and p57Kip2, two proteins modulating various transitions of the cell cycle, appear to play key functions. In this review, the major features of p27 and p57 will be described, focusing, in particular, on their recently identified roles not directly correlated with cell cycle modulation. Then, their possible roles as molecular effectors of polyphenols' activities will be discussed. Polyphenols represent a large family of natural bioactive molecules that have been demonstrated to exhibit promising protective activities against several human diseases. Their use has also been proposed in association with classical therapies for improving their clinical effects and for diminishing their negative side activities. The importance of p27Kip1 and p57Kip2 in polyphenols' cellular effects will be discussed with the aim of identifying novel therapeutic strategies for the treatment of important human diseases, such as cancers, characterized by an altered control of growth.

Effects of Germline VHL Deficiency on Growth, Metabolism, and Mitochondria.

Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222C→A, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598C→T) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).

High Dosage Lithium Treatment Induces DNA Damage and p57Kip2 Decrease.

Lithium salt is the first-line therapeutic option for bipolar disorder and has been proposed as a potential antitumoral drug. The effects of LiCl treatment were investigated in SH-SY5Y, a human neuroblastoma cell line and an in vitro model of dopaminergic neuronal differentiation. LiCl, at the dosage used in psychiatric treatment, does not affect cell proliferation, while at higher doses it delays the SH-SY5Y cell division cycle and for prolonged usage reduces cell viability. Moreover, the ion treatment affects DNA integrity as demonstrated by accumulation of p53 and γH2AX (the phosphorylated form of H2AX histone), two important markers of genome damage. p57Kip2, a CIP/Kip protein, is required for proper neuronal maturation and represents a main factor of response to stress including genotoxicity. We evaluated the effect of lithium on p57Kip2 levels. Unexpectedly, we found that lithium downregulates the level of p57Kip2 in a dose-dependent manner, mainly acting at the transcriptional level. A number of different approaches, mostly based on p57Kip2 content handling, confirmed that the CKI/Kip reduction plays a key role in the DNA damage activated by lithium and suggests the unanticipated view that p57Kip2 might be involved in DNA double-strand break responses. In conclusion, our study identified novel roles for p57Kip2 in the molecular mechanism of lithium at high concentration and, more in general, in the process of DNA repair.

Genetic and Epigenetic Control of CDKN1C Expression: Importance in Cell Commitment and Differentiation, Tissue Homeostasis and Human Diseases.

The CDKN1C gene encodes the p57Kip2 protein which has been identified as the third member of the CIP/Kip family, also including p27Kip1 and p21Cip1. In analogy with these proteins, p57Kip2 is able to bind tightly and inhibit cyclin/cyclin-dependent kinase complexes and, in turn, modulate cell division cycle progression. For a long time, the main function of p57Kip2 has been associated only to correct embryogenesis, since CDKN1C-ablated mice are not vital. Accordingly, it has been demonstrated that CDKN1C alterations cause three human hereditary syndromes, characterized by altered growth rate. Subsequently, the p57Kip2 role in several cell phenotypes has been clearly assessed as well as its down-regulation in human cancers. CDKN1C lies in a genetic locus, 11p15.5, characterized by a remarkable regional imprinting that results in the transcription of only the maternal allele. The control of CDKN1C transcription is also linked to additional mechanisms, including DNA methylation and specific histone methylation/acetylation. Finally, long non-coding RNAs and miRNAs appear to play important roles in controlling p57Kip2 levels. This review mostly represents an appraisal of the available data regarding the control of CDKN1C gene expression. In addition, the structure and function of p57Kip2 protein are briefly described and correlated to human physiology and diseases.

p27Kip1 and human cancers: A reappraisal of a still enigmatic protein.

p27Kip1 is a cell cycle regulator firstly identified as a cyclin-dependent kinase inhibitor. For a long time, its function has been associated to cell cycle progression inhibition at G1/S boundary in response to antiproliferative stimuli. The picture resulted complicated by the discovery that p27Kip1 is an intrinsically unstructured protein, with numerous CDK-dependent and -independent functions and involvement in many cellular processes, such as cytoskeleton dynamics and cell motility control, apoptosis and autophagy activation. Depending on the cell context, these activities might turn to be oncogenic and stimulate cancer progression and metastatization. Nevertheless, p27Kip1 role in cancer biology suppression was underscored by myriad data reporting its down-regulation and/or cytoplasmic relocalization in different tumors, while usually no genetic alterations were found in human cancers, making the protein a non-canonical oncosuppressor. Recently, mostly due to advances in genomic analyses, CDKN1B, p27Kip1 encoding gene, has been found mutated in several cancers, thus leading to a profound reappraisal of CDKN1B role in tumorigenesis. This review summarizes the main p27Kip1 features, with major emphasis to its role in cancer biology and to the importance of CDKN1B mutations in tumor development.

Tyrosine kinase inhibitors and mesenchymal stromal cells: effects on self-renewal, commitment and functions.

The hope of selectively targeting cancer cells by therapy and eradicating definitively malignancies is based on the identification of pathways or metabolisms that clearly distinguish "normal" from "transformed" phenotypes. Some tyrosine kinase activities, specifically unregulated and potently activated in malignant cells, might represent important targets of therapy. Consequently, tyrosine kinase inhibitors (TKIs) might be thought as the "vanguard" of molecularly targeted therapy for human neoplasias. Imatinib and the successive generations of inhibitors of Bcr-Abl1 kinase, represent the major successful examples of TKI use in cancer treatment. Other tyrosine kinases have been selected as targets of therapy, but the efficacy of their inhibition, although evident, is less definite. Two major negative effects exist in this therapeutic strategy and are linked to the specificity of the drugs and to the role of the targeted kinase in non-malignant cells. In this review, we will discuss the data available on the TKIs effects on the metabolism and functions of mesenchymal stromal cells (MSCs). MSCs are widely distributed in human tissues and play key physiological roles; nevertheless, they might be responsible for important pathologies. At present, bone marrow (BM) MSCs have been studied in greater detail, for both embryological origins and functions. The available data are evocative of an unexpected degree of complexity and heterogeneity of BM-MSCs. It is conceivable that this grade of intricacy occurs also in MSCs of other organs. Therefore, in perspective, the negative effects of TKIs on MSCs might represent a critical problem in long-term cancer therapies based on such inhibitors.

Iron overload enhances human mesenchymal stromal cell growth and hampers matrix calcification.

BACKGROUND: Iron overload syndromes include a wide range of diseases frequently associated with increased morbidity and mortality. Several organs are affected in patients with iron overload including liver, heart, joints, endocrine glands, and pancreas. Moreover, severe bone and hemopoietic tissue alterations are observed. Because of the role of bone marrow mesenchymal stromal cells (BM-MSCs) in bone turnover and hematopoiesis, iron effects on primary BM-MSCs cultures were evaluated. METHODS: Primary human BM-MSCs cultures were prepared and the effects of iron on their proliferation and differentiation were characterized by biochemical analyses and functional approaches. RESULTS: Addition of iron to the culture medium strongly increased BM-MSCs proliferation and induced their accelerated S phase entry. Iron enters BM-MSCs through both transferrin-dependent and transferrin-independent mechanisms, inducing the accumulation of cyclins E and A, the decrease of p27(Kip1), and the activation of MAPK pathway. Conversely, neither apoptotic signs nor up-regulation of reactive oxygen species were observed. Iron inhibited both differentiation of BM-MSCs into osteoblasts and in vitro matrix calcification. These effects result from the merging of inhibitory activities on BM-MSCs osteoblastic commitment and on the ordered matrix calcification process. CONCLUSIONS: We demonstrated that BM-MSCs are a target of iron overload. Iron accelerates BM-MSCs proliferation and affects BM-MSCs osteoblastic commitment, hampering matrix calcification. GENERAL SIGNIFICANCE: Our study reports, for the first time, that iron, at concentration found in overloaded patient sera, stimulates the growth of BM-MSCs, the BM multipotent stromal cell component. Moreover, iron modulates the physiological differentiation of these cells, affecting bone turnover and remodeling.

Histone Deacetylase Inhibitors Increase p27(Kip1) by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation.

Histone deacetylase inhibitors (HDACIs) represent an intriguing class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important clinical activities at well tolerated doses. HDACIs also interfere with the aging process and are involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation, and apoptosis. Here, we evaluated the effects of HDACIs on p27(Kip1), a key cyclin-dependent kinase inhibitor (CKI). We observed that HDACI-dependent antiproliferative activity is associated with p27(Kip1) accumulation due to a reduced protein degradation. p27(Kip1) removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27(Kip1) stability through downregulation of Skp2 protein levels. Skp2 decline is only partially due to a reduced Skp2 gene expression. Conversely, the protein decrease is more profound and enduring compared to the changes of Skp2 transcript. This argues for HDACIs effects on Skp2 protein posttranslational modifications and/or on its removal. In summary, we demonstrate that HDACIs increase p27(Kip1) by hampering its nuclear ubiquitination/degradation. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities.

Pyrosequencing evaluation of low-frequency KRAS mutant alleles for EGF receptor therapy selection in metastatic colorectal carcinoma.

AIM: To evaluate whether pyrosequencing (PS) improves the KRAS mutational status predictive value. PATIENTS & METHODS: A retrospective analysis of KRAS mutations by PS and direct sequencing (DS) in 192 metastatic colorectal carcinomas (mCRCs), subgrouped in 51 KRAS mutated at PS and 141 KRAS wild-type at DS. RESULTS: DS failed to detect low-frequency KRAS mutations in four out of 51 mCRCs, whereas PS detected 12 additional low-frequency KRAS mutations in 141 mCRCs KRAS wild-type at DS. After reanalyzing by PS 97 KRAS wild-type tumors treated with anti-EGF receptor (EGFR) antibodies, nine additional mutations were revealed in nonresponders, whereas none of responders exhibited a KRAS-mutated genotype. Of note, KRAS-mutated tumors upon PS showed a worst progression-free survival after EGFR therapy. Finally, PS allowed the detection of additional NRAS, BRAF and exon 20 PIK3CA mutations mostly in KRAS wild-type mCRCs resistant to EGFR therapy. CONCLUSION: PS detection of low-frequency mutations may improve the KRAS predictive value for EGFR therapy selection.