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A group of functionalized fluorene derivatives that are structurally similar to the cellular prion protein ligand N , N '-(methylenedi-4,1-phenylene)bis [2-(1-pyrrolidinyl)acetamide] (GN8) have been synthesized. These compounds show remarkable native fluorescence due to the fluorene ring. The substituents introduced at positions 2 and 7 of the fluorene moiety are sufficiently flexible to accommodate the beta-conformational folding that develops in amyloidogenic proteins. Changes in the native fluorescence of these fluorene derivatives provide evidence of transformations in the amyloidogenic aggregation processes of insulin. The increase observed in the fluorescence intensity of the sensors in the presence of native insulin or amyloid aggregates suggest their potential use as fluorescence probes for detecting abnormal conformations; therefore, the compounds can be proposed for use as "turn-on" fluorescence sensors. Protein-sensor dissociation constants are in the 5-10 μM range and an intermolecular charge transfer process between the protein and the sensors can be successfully exploited for the sensitive detection of abnormal insulin conformations. The values obtained for the Stern-Volmer quenching constant for compound 4 as a consequence of the sensor-protein interaction are comparable to those obtained for the reference compound GN8. Fluorene derivatives showed good performance in scavenging reactive oxygen species (ROS), and they show antioxidant capacity according to the FRAP and DPPH assays.

The functional relevance of K + and Ca 2+ ion channels in the "Store Operated Calcium Entry" (SOCE) during B and T lymphocyte activation is well proven. However, their role in the process of T- and B- cell development and selection is still poorly defined. In this scenario, our aim was to characterize the expression of the ether à-go-go-related gene 1 (ERG1) and K V 1.3 K + channels during the early stages of mouse lymphopoiesis and analyze how they affect Ca 2+ signaling, or other signaling pathways, known to mediate selection and differentiation processes of lymphoid clones. We provide here evidence that the mouse (m)ERG1 is expressed in primary lymphoid organs, bone marrow (BM), and thymus of C57BL/6 and SV129 mice. This expression is particularly evident in the BM during the developmental stages of B cells, before the positive selection (large and small PreB). mERG1 is also expressed in all thymic subsets of both strains, when lymphocyte positive and negative selection occurs. Partially overlapping results were obtained for K V 1.3 expression. mERG1 and KV1.3 were expressed at significantly higher levels in B-cell precursors of mice developing an experimental autoimmune encephalomyelitis (EAE). The pharmacological blockage of ERG1 channels with E4031 produced a significant reduction in intracellular Ca 2+ after lymphocyte stimulation in the CD4 + and double-positive T-cell precursors' subsets. This suggests that ERG1 might contribute to maintaining the electrochemical gradient responsible for driving Ca 2+ entry, during T-cell receptor signaling which sustains lymphocyte selection checkpoints. Such role mirrors that performed by the shaker-type K V 1.3 potassium channel during the activation process of mature lymphocytes. No effects on Ca 2+ signaling were observed either in B-cell precursors after blocking K V 1.3 with PSORA-4. In the BM, the pharmacological blockage of ERG1 channels produced an increase in ERK phosphorylation, suggesting an effect of ERG1 in regulating B-lymphocyte precursor clones' proliferation and checkpoint escape. Overall, our results suggest a novel physiological function of ERG1 in the processes of differentiation and selection of lymphoid precursors, paving the way to further studies aimed at defining the expression and role of ERG1 channels in immune-based pathologies in addition to that during lymphocyte neoplastic transformation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sala, Staderini, Lottini, Duranti, Angelini, Constantin and Arcangeli.)

Implantable electrochemical sensors that enable the real-time detection of significant biomarkers offer huge potential for the enhancement and personalisation of therapies; however, biofouling is a key challenge encountered by any implantable system. This is particularly an issue immediately after implantation, when the foreign body response and associated biofouling processes are at their most active in passivating a foreign object. Here, we present the development of a sensor protection and activation strategy against biofouling, based on coatings consisting of a pH-triggered, dissolvable polymer, that covered a functionalised electrode surface. We demonstrate that reproducible delayed sensor activation can be achieved, and that the length of this delay can be controlled by the optimisation of coating thickness, homogeneity and density through tuning of the coating method and temperature. Comparative evaluation of the polymer-coated and uncoated probe-modified electrodes in biological media revealed significant improvements in their anti-biofouling characteristics, demonstrating that this offers a promising approach to the design of enhanced sensing devices.

Introduction: Carfilzomib, a potent, irreversible, selective proteasome inhibitor has demonstrated consistent results in relapsed/refractory multiple myeloma (RRMM) combined with lenalidomide and dexamethasone (KRd). No prospective studies are yet available that analyzed the efficacy of the KRd combination., Methods: Herein, we report a multicenter prospective observational study on 85 patients who were treated with KRd combination as the second or third line of treatment, according to standard practice., Results: The median age was 61 years; high-risk cytogenetic was found in 26% and renal impairment (estimated glomerular filtration rate (eGFR) <60 ml/min) in 17%. After a median follow-up of 40 months, patients received a median number of 16 cycles of KRd, with a median duration of treatment (DoT) of 18 months (range, 16.1-19.2 months). The overall response rate was 95%, with a high-quality response (≥very good partial remission [VGPR]) in 57% of the patients. The median progression-free survival (PFS) was 36 months (range, 29.1-43.2 months). Achievement of at least VGPR and a previous autologous stem cell transplantation (ASCT) were associated with longer PFS. The median overall survival (OS) was not reached (NR); the 5-year OS rate was 73%. Nineteen patients underwent KRd treatment as a bridge to autologous transplantation, obtaining a post-transplant minimal residual disease (MRD) negativity in 65% of cases. The most common adverse events were hematological, followed by infection and cardiovascular events, rarely G3 or higher, with a discontinuation rate for toxicities of 6%. Our data confirmed the feasibility and safety of the KRd regimen in real life., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Antonioli, Pilerci, Attucci, Buda, Gozzetti, Candi, Simonetti, Giudice, Ciofini, Staderini, Grammatico, Buzzichelli, Messeri, Bocchia, Galimberti and Vannucchi.)

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrP Sc ) of the cellular prion protein (PrP C ). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrP C conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrP Sc . Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrP C levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrP Sc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The development of robust implantable sensors is important in the successful advancement of personalised medicine as they have the potential to provide in situ real-time data regarding the status of health and disease and the effectiveness of treatment. Tissue pH is a key physiological parameter and herein, we report the design, fabrication, functionalisation, encapsulation and protection of a miniaturised, self-contained, electrochemical pH sensor system and characterisation of sensor performance. Notably for the first time in this environment the pH sensor was based on a methylene blue redox reporter which showed remarkable robustness, accuracy and sensitivity. This was achieved by encapsulation of a self-assembled monolayer containing methylene blue entrapped within a Nafion layer. Another powerful feature was the incorporation, within the same implanted device, of a fabricated on-chip Ag/AgCl reference electrode - vital in any electrochemical sensor, but often ignored. When utilised in vivo, the sensor allowed accurate tracking of externally induced pH changes within a naturally occurring ovine lung cancer model, and correlated well with single point laboratory measurements made on extracted arterial blood, whilst enabling in vivo time-dependent measurements. The sensors functioned robustly whilst implanted, and maintained in vitro function once extracted and together, these results demonstrate proof-of-concept of the ability to sense real-time intratumoral tissue pH changes in vivo., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: Currently, the regimen with bortezomib plus melphalan and prednisone (VMP) is a standard treatment for multiple myeloma and it is recommended as the first-line therapy for patients with multiple myeloma (MM) ineligible for high-dose chemotherapy with autologous stem-cell transplantation. Objectives: Participants of the clinical trial are highly selected populations; therefore, the aim of this study was to present observations from real practice that can provide important information for practitioners and to investigate clinical outcomes of VMP regimen in elderly patients with newly diagnosed MM. Patients and Methods: We retrospectively analyzed the data on the efficacy and survival parameters, such as overall survival (OS) and event-free survival (EFS), with attention to the effect of gender, age and International Staging System (ISS) stage, of VMP regimen in 164 patients with newly diagnosed MM not eligible for high-dose chemotherapy with autologous stem-cell transplantation (median age, 75 years; range, 60-86 years). Results: Patients aged 75 years or older constituted 50.6% of the study cohort. Frail patients were 10.36%, according to the clinical frailty scale of geriatric assessment (GA). A total of 1203 courses of VMP regimen (mainly VMP 1-29, 99.16 %) were administered. The median cumulative delivered dose of bortezomib was 46.8 mg/m2. The overall response rate (ORR), including all patients with a partial response or better, was 81.7% and the complete response rate (CRR) was 10.36 %. After a median 38.51 months of follow-up, the median overall survival (OS) was 34.33 months; the median event-free survival (EFS) after VMP and second-line therapy (mainly Rd, 56.31%) were 18.51 and 10.75 months, respectively. In the subgroup of patients with 75 years or older the median OS was 29.76 months; the median EFS after first and second-line therapy were 17.76 and 8.93 months, respectively. The hazard ratio for OS was 2.276 ( p -value 0.046) and for EFS was 1.507 ( p -value 0.055) for the ISS stage II and III group. Age and gender were not negative predictors of survival. Conclusions: VMP treatment is highly effective in the first-line therapy of elderly patients with multiple myeloma ineligible for HDT with auto-SCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Buda, Del Giudice, Antonioli, Ghio, Orciuolo, Morganti, Martini, Staderini, Galimberti and Petrini.)

Carfilzomib-lenalidomide-dexamethasone (KRd) has been approved for the treatment of relapsed/refractory multiple myeloma (RRMM). We conducted a retrospective analysis of 197 RRMM patients (pts) between January 2016 and March 2018 in six Italian hematologic centers, with the aim to evaluate efficacy and safety of KRd in real-life. At KRd initiation 27% carried high risk cytogenetic abnormalities (HRCA) [del17p and/or t(4;14) and/or t(14;16)], median number of prior lines of therapy was 2 (1-8), nearly all pts (96%) received prior bortezomib (18% refractory) while 45% were exposed to lenalidomide (R; 22% refractory). At the median of 12.5 months, 52% of the pts had discontinued treatment, mainly (66%) for progression. Main grade 3-4 adverse events were neutropenia (21%), infections (11%), and hypertension (6%). Overall, the response rate was 88%. The median progression-free survival (PFS) was 19.8 months and 1-year overall survival (OS) rate was 80.6%. By subgroup analysis, extended PFS and OS were observed for pts who received ≤2 prior lines of therapy (HR = 0.42, p < 0.001 and HR = 0.35, p = 0.001, respectively), not refractory to prior R (HR = 0.37, p < 0.001, and HR = 0.47, p = 0.024), without HRCA (HR = 0.33, p = 0.005 and HR = 0.26, p = 0.016) and achieving ≥ very good partial response (VGPR; HR = 0.17, p < 0.001 and HR = 0.18, p < 0.001). In conclusion, KRd demonstrated to be effective in RRMM pts treated in real-world setting, without new safety concerns. Better survival outcomes emerged for pts with ≤2 prior lines of therapy, achieving at least a VGPR, and without HRCA., (© 2020 John Wiley & Sons Ltd.)

Here we report the synthesis of a novel methylene blue-polymyxin conjugate and demonstrate its light-mediated killing of Gram-negative bacteria on skin models of infection demonstrating a 10 8 decrease in bacterial colony-forming units.

Proteases are ideal target biomarkers as they have been implicated in many disease states, including steps associated with cancer progression. Electrochemical peptide-based biosensors have attracted much interest in recent years. However, the significantly large size of the electrodes typically used in most of these platforms has led to performance limitations. These could be addressed by the enhancements offered by microelectrodes, such as rapid response times, improved mass transport, higher signal-to-noise and sensitivity, as well as more localised and less invasive measurements. We present the production and characterisation of a miniaturised electrochemical biosensor for the detection of trypsin, based on 25 μm diameter Pt microelectrodes (rather than the ubiquitous Au electrodes), benchmarked by establishing the equivalent Pt macroelectrode response in terms of quantitative response to the protease, the kinetics of cleavage and the effects of non-specific protein binding and temperature. Interestingly, although there was little difference between Au and Pt macroelectrode response, significant differences were observed between the responses of the Pt macroelectrode and microelectrode systems indicative of increased reproducibility in the microelectrode SAM structure and sensor performance between the electrodes, increased storage stability and a decrease in the cleavage rate at functionalised microelectrodes, which is mitigated by measurement at normal body temperature. Together, these results demonstrate the robustness and sensitivity of the miniaturised sensing platform and its ability to operate within the clinically-relevant concentration ranges of proteases in normal and disease states. These are critical features for its translation into implantable devices.

Objectives: ASCT is currently the "gold standard" first-line treatment for multiple myeloma patients younger than 65 years old, and limited data on efficacy and safety in older patients are available., Methods: We retrospectively analyzed a cohort of 83 newly diagnosed multiple myeloma patients aged 65 or older. All patients were evaluated for fitness at diagnosis and after bortezomib-based induction treatment., Results and Conclusions: All patients collected an adequate PBSC graft, mainly after G-CSF plus cyclophosphamide; a median of 6.47 × 10 6 /kg CD34 + cells was collected. The conditioning regimen consisted of melphalan 100, 140 and 200 mg/m 2 in 40, 15 and 28 patients, respectively. Median time to neutrophils' and platelets' recovery was 11 and 12 days, respectively. Adverse events of any grade were referred by 40% of patients. The overall response rate was 93%, CR/sCR were 39%. Median PFS was 35 months; median OS was not reached. In our study cohort, the achievement of at least VGPR after induction therapy and the obtainment of CR/sCR after ASCT are the only parameters that were associated with an improved PFS. ASCT is an effective and safe first-line treatment approach, a careful patients selection reduce the toxicity of the procedure., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A new class of quinoline derivatives, bearing amino chains at C-4 and a styryl group at C-2, were tested on Leishmania donovani promastigotes and axenic and intracellular Leishmania pifanoi amastigotes. The introduction of the C-4 substituent improves the activity, which is due to interference with the mitochondrial activity of the parasite and its concomitant bioenergetic collapse by ATP exhaustion. Some compounds show a promising antileishmanial profile, with low micromolar or submicromolar activity on promastigote and amastigote forms and a good selectivity index., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Human neutrophil elastase (HNE) is a serine protease, produced by polymorphonuclear neutrophils (PMNs), whose uncontrolled production has been associated with various inflammatory disease states as well as tumour proliferation and metastasis. Here we report the development and characterisation of an electrochemical peptide-based biosensor, which enables the detection of clinically relevant levels of HNE. The sensing platform was characterised in terms of its analytical performance, enzymatic cleavage kinetics and cross-reactivity and applied to the quantitative detection of protease activity from PMNs from human blood., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The term electroceutical has been used to describe implanted devices that deliver electrical stimuli to modify biological function. Herein, we describe a new concept in electroceuticals, demonstrating for the first time the electrochemical activation of metal-based prodrugs. This is illustrated by the controlled activation of Pt(iv) prodrugs into their active Pt(ii) forms within a cellular context allowing selectivity and control of where, when and how much active drug is generated.

Optical medical imaging is a rapidly growing area of research and development that offers a multitude of healthcare solutions both diagnostically and therapeutically. In this review, some of the most recently described peptide-based optical probes are reviewed with a special emphasis on their in vivo use and potential application in a clinical setting., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The vinyl ether benzyloxycarbonyl (VeZ) protecting group is selectively cleaved by treatment with tetrazines via an inverse electron-demand Diels-Alder reaction. This represents a new orthogonal protecting group for solid-phase peptide synthesis, with Fmoc-Lys(VeZ)-OH as a versatile alternative to Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH, as demonstrated by the synthesis of two biologically relevant cyclic peptides.

Multiple myeloma (MM) is characterized, in about 80% of cases, by the production of monoclonal intact immunoglobulin and more than 95% of them have elevated concentrations of involved (i.e. of the same class of intact immunoglobulin) free light chain (FLC). The introduction of novel therapeutic strategies has changed the natural history of the disease, leading to new manifestations of relapse. Light chain escape (LCE) is a pattern of relapse in which the FLC increase is not accompanied by a concomitant raise of the original monoclonal component (MC). Here we present a case of a 55-year-old man with an IgG kappa MM stage III diagnosed in September 2007. At presentation an IgG kappa MC and urine Bence Jones protein (BJP) kappa were present. Bone marrow biopsy (BMB) showed the presence of 80% monotypic kappa plasma cells (PCs). The patient received bortezomib, thalidomide, dexamethasone before undergoing a double autologous stem cell transplantation (ASCT) in October 2008 and April 2009. In May 2011 he relapsed showing the same pattern of presentation and treatment with lenalidomide and dexamethasone was started. ln May 2013 serum and urine immunofixation and FLC became negative. In September 2014, an increase of kappa FLC was observed, while serum and urine immunofixations remained negative until January 2015, when urine immunofixation became positive. Eventually, in February 2015, serum immunofixation revealed the presence of a free kappa MC. After a new BMB showing 80% of monotypic kappa PCs, a LCE relapse was diagnosed and the patient started the treatment with bendamustine, bortezomib and dexamethasone. In the present case, the increase of kappa FLC has indicated relapse 4 and 5 months earlier than urine and serum IFE, respectively. Our observation confirms that it is advisable to routinely perform FLC or BJP during follow up of MM patients undergoing ASCT and/or treatment with biological drugs to ensure that LCE is not missed.

Brain amyloid depositions are the main hallmarks of Alzheimer's and other protein misfolding diseases. Since they are believed to precede clinical symptoms by several years, imaging of such fibrillar aggregates is particularly suitable to diagnose the onset of the disease in its early stage and monitor its progression. In this context, near infrared (NIR) imaging has been proposed as a promising and non-invasive method to visualize amyloid plaques in vivo because of its acceptable depth of penetration and minimal degree of tissue damage. In this tutorial review, we describe the main chemical and physicochemical features of probes associated with fluorescence emission in the NIR region. The review focuses on the recent progress and improvements in the development of small-molecule NIR fluorescent probes and their in vivo application in living animals. In addition, the possible therapeutic application of NIR probes to block the pathological aggregation process will be discussed, raising the fascinating possibility of their exploitation as theranostic agents.

The introduction of immunomodulatory drugs such as lenalidomide combined with dexamethasone (Len/Dex) has improved the outcome of patients with relapsed/refractory multiple myeloma (RRMM). Few data are currently available which investigate whether paraprotein relapse represents an indication for starting a new treatment. The aim of our retrospective, single-center study was to analyze the impact of disease status (relapsed/refractory) and type of relapse (clinical/paraprotein) on response rate and time-to-next-treatment (TNT). We included 74 patients (median age 70 years) with RRMM treated with Len/Dex until progression or unacceptable toxicity from 2008 to 2012. Age and disease status were not factors affecting overall response rate (ORR) and median TNT, but TNT was significantly longer in patients with asymptomatic compared to clinical relapse (34 vs. 19 months, p<0.008). In conclusion, Len/Dex represents an effective treatment with satisfactory ORR and outcomes in RRMM, especially for patients starting therapy in asymptomatic relapse.

Prion diseases (PrDs) are fatal neurodegenerative disorders, for which no effective therapeutic and diagnostic tools exist. The main pathogenic event has been identified as the misfolding of a disease-associated prion protein. Nevertheless, pathogenesis seems to involve an intricate array of concomitant processes. Thus, it may be unlikely that drugs acting on single targets can effectively control PrDs. In addition, diagnosis occurs late in the disease process, by which point it is difficult to determine a successful therapeutic intervention. In this context, multitarget ligands (MTLs) and theranostic ligands (TLs) emerge for their potential to effectively cure and diagnose PrDs. In this review, we discuss the medicinal chemistry challenges of identifying novel MTLs and TLs against PrDs, and envision their impact on prion drug discovery.

Prion diseases are fatal neurodegenerative disorders that affect humans and animals and for which no pharmacological treatment is available. Compounds consisting of two identical moieties joined via an appropriate spacer (i.e. bivalent compounds) have turned out to be effective tools to prevent prion fibril formation and exhibit an improved biological profile with regard to the corresponding monovalent derivatives. In this review we discuss the importance of the bivalent strategy as a viable approach to design new chemical entities to combat prion diseases.

(E)-6-Methyl-4'-amino-2-styrylquinoline (3) is a small molecule with the proper features to potentially diagnose, deliver therapy and monitor response to therapy in protein misfolding diseases. These features include compound fluorescent emission in the NIR region and its ability to interact with both Aβ and prion fibrils, staining them with high selectivity. Styrylquinoline 3 also inhibits Aβ self-aggregation in vitro and prion replication in the submicromolar range in a cellular context. Furthermore, it is not toxic and is able to cross the blood brain barrier in vitro (PAMPA test).

Prion diseases are fatal neurodegenerative and infectious disorders for which effective pharmacological tools are not yet available. This unmet challenge and the recently proposed interplay between prion diseases and Alzheimer's have led to a more urgent demand for new antiprion agents. Herein, we report the identification of a novel bifunctional diketopiperazine (DKP) derivative 1 d, which exhibits activity in the low micromolar range against prion replication in ScGT1 cells, while showing low cytotoxicity. Supported by properly addressed molecular modeling studies, we hypothesized that a planar conformation is the major determinant for activity in this class of compounds. Moreover, studies aimed at assessing the mechanism-of-action at the molecular level showed that 1 d might interact directly with recombinant prion protein (recPrP) to prevent its conversion to the pathogenic misfolded prion protein (PrP(Sc))-like form. This investigation suggests that DKP based antiprion compounds can serve as a promising lead scaffold in developing new drugs to combat prion diseases.