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Multiple myeloma (MM) is linked to chronic NF-κB activity in myeloma cells, but this activity is generally considered a cell-autonomous property of the cancer cells. The precise extent of NF-κB activation and the contributions of the physical microenvironment and of cell-to-cell communications remain largely unknown. By quantitative immunofluorescence, we found that NF-κB is mildly and heterogeneously activated in a fraction of MM cells in human BMs, while only a minority of MM cells shows a strong activation. To gain quantitative insights on NF-κB activation in living MM cells, we combined advanced live imaging of endogenous p65 Venus-knocked-in in MM.1S and HS-5 cell lines to model MM and mesenchymal stromal cells (MSCs), cell co-cultures, microfluidics and custom microbioreactors to mimic the 3D-interactions within the bone marrow (BM) microenvironment. We found that i) reciprocal MM-MSC paracrine crosstalk and cell-to-scaffold interactions shape the inflammatory response in the BM; ii) the pro-inflammatory cytokine IL-1β, abundant in MM patients' plasma, activates MSCs, whose paracrine signals are responsible for strong NF-κB activation in a minority of MM cells; iii) IL-1β, but not TNF-α, activates NF-κB in vivo in BM-engrafted MM cells, while its receptor inhibitor Anakinra reduces the global NF-κB activation. We propose that NF-κB activation in the BM of MM patients is mild, restricted to a minority of cells and modulated by the interplay of restraining physical microenvironmental cues and activating IL-1β-dependent stroma-to-MM crosstalk., (© 2024. The Author(s).)

Background and Aims: Ultrasound nerve cross-sectional area (CSA) of patients affected with axonal neuropathy usually shows normal value. Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) seems to represent an exception, showing smaller CSA, but previous reports did not test for biallelic RFC1 gene repeat expansions., Methods: We compared nerve CSA from CANVAS patients (tested positive for biallelic RFC1 gene repeat expansions) with the CSA from a group of patients with chronic idiopathic axonal polyneuropathy (CIAP) who tested negative for RFC1 gene repeat expansions, hereditary axonal neuropathy (Charcot-Marie-Tooth type 2, CMT2), and Friedreich ataxia (FRDA)., Results: We enrolled 15 CANVAS patients (eight men, mean age 66.3 ± 11.5 years, mean disease duration 9.3 ± 4.1 years), affected with sensory axonal neuronopathy. Controls consisted of 13 CIAP (mean age 68.5 ± 12.8 years, seven men), seven CMT2 (mean age 47.9 ± 18.1 years, four men), 12 FRDA (mean age 33.7 ± 8.8, five men). Nerve ultrasound was performed at median, ulnar, sciatic, sural, and tibial nerves and brachial plexus, bilaterally. The nerve CSA from CANVAS patients was significantly smaller than the one from the other cohorts at several sites with significant and high accuracy at Receiver-operating characteristic (ROC) curve analyses. RFC1 AAGGG pentanucleotide expansion, disease duration, and disability did not correlate with CSA at any site, after Bonferroni correction., Interpretation: Decreased sonographic nerve sizes, in arms and legs, in patients with sensory neuropathy and normal motor conduction studies could point to CANVAS-spectrum disease and help guide appropriate genetic testing., (© 2024 Peripheral Nerve Society.)

A "watch and wait" strategy, delaying treatment until active disease manifests, is adopted for most CLL cases; however, prognostic models incorporating biomarkers have shown to be useful to predict treatment requirement. In our prospective O-CLL1 study including 224 patients, we investigated the predictive role of 513 microRNAs (miRNAs) on time to first treatment (TTFT). In the context of this study, six well-established variables (i.e., Rai stage, beta-2-microglobulin levels, IGVH mutational status, del11q, del17p, and NOTCH1 mutations) maintained significant associations with TTFT in a basic multivariable model, collectively yielding a Harrell's C-index of 75% and explaining 45.4% of the variance in the prediction of TTFT. Concerning miRNAs, 73 out of 513 were significantly associated with TTFT in a univariable model; of these, 16 retained an independent relationship with the outcome in a multivariable analysis. For 8 of these (i.e., miR-582-3p, miR-33a-3p, miR-516a-5p, miR-99a-5p, and miR-296-3p, miR-502-5p, miR-625-5p, and miR-29c-3p), a lower expression correlated with a shorter TTFT, whereas in the remaining eight (i.e., miR-150-5p, miR-148a-3p, miR-28-5p, miR-144-5p, miR-671-5p, miR-1-3p, miR-193a-3p, and miR-124-3p), the higher expression was associated with shorter TTFT. Integrating these miRNAs into the basic model significantly enhanced predictive accuracy, raising the Harrell's C-index to 81.1% and the explained variation in TTFT to 63.3%. Moreover, the inclusion of the miRNA scores enhanced the integrated discrimination improvement (IDI) and the net reclassification index (NRI), underscoring the potential of miRNAs to refine CLL prognostic models and providing insights for clinical decision-making. In silico analyses on the differently expressed miRNAs revealed their potential regulatory functions of several pathways, including those involved in the therapeutic responses. To add a biological context to the clinical evidence, an miRNA-mRNA correlation analysis revealed at least one significant negative correlation between 15 of the identified miRNAs and a set of 50 artificial intelligence (AI)-selected genes, previously identified by us as relevant for TTFT prediction in the same cohort of CLL patients. In conclusion, the identification of specific miRNAs as predictors of TTFT holds promise for enhancing risk stratification in CLL to predict therapeutic needs. However, further validation studies and in-depth functional analyses are required to confirm the robustness of these observations and to facilitate their translation into meaningful clinical utility.

Background : The necessity of using central venous catheters (CVCs) in hemodialysis, coupled with their associated complications, remains a critical concern in nephrology. This study aims to compare the short-term prognosis of tunneled (T-CVC) and non-tunneled (NT-CVC) CVCs in acute hemodialysis patients, specifically focusing on infection rates, malpositioning, and lumen thrombosis within the first three weeks post-insertion. Methods : A retrospective analysis was conducted on 176 CVCs placed between January and December 2023 at the Policlinico di Modena and the Ospedale Civile di Baggiovara. Patient demographics, CHA2DS2-VASc scores, and comorbid conditions were recorded at the time of catheter placement. Outcomes assessed included catheter-related infections, malpositioning, and lumen thrombosis. Statistical analyses, including Chi-square tests, Fisher's exact tests, and Kaplan-Meier survival analysis, were performed to evaluate differences between T-CVCs and NT-CVCs. Results : The sample comprised 43% females with a mean age of 69.3 years (SD 13.9) and a mean CHADS-VASC score of 3.72 (SD 1.4). Hypertension (90%) was the most prevalent comorbidity. Of the 176 CVCs, 127 were T-CVCs and 49 were NT-CVCs. Infection rates were 3.15% for T-CVCs and 8.16% for NT-CVCs ( p = 0.07). Malpositioning occurred in 0.79% of T-CVCs and 4.08% of NT-CVCs ( p = 0.47). There was one case of lumen thrombosis in the NT-CVC group. Kaplan-Meier analysis indicated a significant divergence in infection-related catheter survival favoring T-CVCs after ten days ( p = 0.034). Conclusions : While non-tunneled CVCs do not significantly alter short-term prognosis compared to tunneled CVCs, the latter show a better infection-related survival rate beyond ten days. Therefore, primary insertion of T-CVCs may be preferable when resources and clinical conditions permit, although NT-CVCs remain a viable option when immediate T-CVC insertion is challenging.

Background: We aimed to investigate the clinical features of a large cohort of patients with myelin protein zero ( MPZ )-related neuropathy, focusing on the five main mutation clusters across Italy., Methods: We retrospectively gathered a minimal data set of clinical information in a series of patients with these frequent mutations recruited among Italian Charcot-Marie-Tooth (CMT) registry centres, including disease onset/severity (CMTES-CMT Examination Score), motor/sensory symptoms and use of orthotics/aids., Results: We collected data from 186 patients: 60 had the p.Ser78Leu variant ('classical' CMT1B; from Eastern Sicily), 42 the p.Pro70Ser (CMT2I; mainly from Lombardy), 38 the p.Thr124Met (CMT2J; from Veneto), 25 the p.Ser44Phe (CMT2I; from Sardinia) and 21 the p.Asp104ThrfsX13 (mild CMT1B; from Apulia) mutation. Disease severity (CMTES) was higher (p<0.001) in late-onset axonal forms (p.Thr124Met=9.2±6.6; p.Ser44Phe=7.8±5.7; p.Pro70Ser=7.6±4.8) compared with p.Ser78Leu (6.1±3.5) patients. Disease progression (ΔCMTES/year) was faster in the p.Pro70Ser cohort (0.8±1.0), followed by p.Ser44Phe (0.7±0.4), p.Thr124Met (0.4±0.5) and p.Ser78Leu (0.2±0.4) patients. Disease severity (CMTES=1.2±1.5), progression (ΔCMTES/year=0.1±0.4) and motor involvement were almost negligible in p.Asp104ThrfsX13 patients, who, however, frequently (78%, p<0.001) complained of neuropathic pain. In the other four clusters, walking difficulties were reported by 69-85% of patients, while orthotic and walking aids use ranged between 40-62% and 16-28%, respectively., Conclusions: This is the largest MPZ (and late-onset CMT2) cohort ever collected, reporting clinical features and disease progression of 186 patients from five different clusters across Italy. Our findings corroborate the importance of differentiating between 'classical' childhood-onset demyelinating, late-onset axonal and mild MPZ -related neuropathy, characterised by different pathomechanisms, in view of different therapeutic targets., Competing Interests: Competing interests: GMF acknowledges donations from Pfizer to support research activities of his Research Unit, financial support from Akcea, Kedrion, Pfizer for participation in national and international meetings and from Akcea, Alnylam and Pharnext for participation in Advisory Boards; MG acknowledges donations from Sanofi Genzyme to support research activities of her Research Unit, financial support from Alnylam and Sanofi Genzyme for participation in national and international Meetings, participation in Advisory Board of Pfizer, speaker honorarium from Sanofi Genzyme; AM acknowledges financial support from Pfizer, Alnylam and Akcea for participation in national and international meetings, participation in Advisory Board of Pfizer, Alnylam and Akcea; DP acknowledges participation in Advisory Board of Inflectis, Alnylam, Akcea, Arvinas, Augustine Tx, DTx; ST is supported by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) - A multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022). AB, LG, TC, PS, MR, SM, YMF, EM, FT, AG, GO, MF, EC, LC, PM, FB, SM, FT, SCP, AS, FM, CP report no disclosure., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Cell-free nucleic acids (cf-NAs) represent a promising biomarker of various pathological and physiological conditions. Since its discovery in 1948, cf-NAs gained prognostic value in oncology, immunology, and other relevant fields. In peritoneal dialysis (PD), blood purification is performed by exposing the peritoneal membrane. Relevant sections: Complications of PD such as acute peritonitis and peritoneal membrane aging are often critical in PD patient management. In this review, we focused on bacterial DNA, cell-free DNA, mitochondrial DNA (mtDNA), microRNA (miRNA), and their potential uses as biomarkers for monitoring PD and its complications. For instance, the isolation of bacterial DNA in early acute peritonitis allows bacterial identification and subsequent therapy implementation. Cell-free DNA in peritoneal dialysis effluent (PDE) represents a marker of stress of the peritoneal membrane in both acute and chronic PD complications. Moreover, miRNA are promising hallmarks of peritoneal membrane remodeling and aging, even before its manifestation. In this scenario, with multiple cytokines involved, mtDNA could be considered equally meaningful to determine tissue inflammation., Conclusions: This review explores the relevance of cf-NAs in PD, demonstrating its promising role for both diagnosis and treatment. Further studies are necessary to implement the use of cf-NAs in PD clinical practice.

Background and Purpose: Biallelic mutation/expansion of the gene RFC1 has been described in association with a spectrum of manifestations ranging from isolated sensory neuro(no)pathy to a complex presentation as cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS). Our aim was to define the frequency and characteristics of small fiber neuropathy (SFN) in RFC1 disease at different stages., Methods: RFC1 cases were screened for SFN using the Neuropathic Pain Symptom Inventory and Composite Autonomic Symptom Score 31 questionnaires. Clinical data were retrospectively collected. If available, lower limb skin biopsy samples were evaluated for somatic epidermal and autonomic subepidermal structure innervation and compared to healthy controls (HCs)., Results: Forty patients, median age at onset 54 years (interquartile range [IQR] 49-61) and disease duration 10 years (IQR 6-16), were enrolled. Mild-to-moderate positive symptoms (median Neuropathic Pain Symptom Inventory score 12.1/50, IQR 5.5-22.3) and relevant autonomic disturbances (median Composite Autonomic Symptom Score 31 37.0/100, IQR 17.7-44.3) were frequently reported and showed scarce correlation with disease duration. A non-length-dependent impairment in nociception was evident in both clinical and paraclinical investigations. An extreme somatic denervation was observed in all patients at both proximal (fibers/mm, RFC1 cases 0.0 vs. HCs 20.5, p < 0.0001) and distal sites (fibers/mm, RFC1 cases 0.0 vs. HCs 13.1, p < 0.0001); instead only a slight decrease was observed in cholinergic and adrenergic innervation of autonomic structures., Conclusions: RFC1 disease is characterized by a severe and widespread somatic SFN. Skin denervation may potentially represent the earliest feature and drive towards the suspicion of this disorder., (© 2023 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

In hadrontherapy centers, ion beams are accelerated by a cyclotron or synchrotron through electric and magnetic fields and, once the treatment energy is reached, ions interact with the patient. The interaction of the beam with magnets, other elements of particle accelerator or with the patient generates secondary radiation and the calculation for the design of the shielding is required. A first assessment of the shielding, generally made by concrete, can be performed by using Monte Carlo simulations, in which neutron yields and the ambient dose equivalent, H*(10), spatial distribution are obtained. The aim of this work is to evaluate, with a Monte Carlo approach, ambient dose equivalent values at various concrete depths generated by the secondary radiation. Simulations are performed considering helium, lithium, carbon, oxygen and iron primary beams. For existing facilities that already accelerates carbon ion, an equivalent carbon ratio is introduced to estimate the ion currents to get, in a given point, the same ambient dose equivalent as a carbon ion. Numerical results have been obtained simulating an ion beam that impinges on a thick iron or international commission on radiation units and measurements (ICRU) tissue, respectively, representing magnets and patient. The secondary radiation is transported through a large concrete shielding where ambient dose equivalent values are calculated as function of concrete thickness., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Trained immunity (TI) is a de facto memory program of innate immune cells, characterized by immunometabolic and epigenetic changes sustaining enhanced production of cytokines. TI evolved as a protective mechanism against infections; however, inappropriate activation can cause detrimental inflammation and might be implicated in the pathogenesis of chronic inflammatory diseases. In this study, we investigated the role of TI in the pathogenesis of giant cell arteritis (GCA), a large-vessel vasculitis characterized by aberrant macrophage activation and excess cytokine production., Methods: Monocytes from GCA patients and from age- and sex-matched healthy donors were subjected to polyfunctional studies, including cytokine production assays at baseline and following stimulation, intracellular metabolomics, chromatin immunoprecipitation-qPCR, and combined ATAC/RNA sequencing. Immunometabolic activation (i.e. glycolysis) was assessed in inflamed vessels of GCA patients with FDG-PET and immunohistochemistry (IHC), and the role of this pathway in sustaining cytokine production was confirmed with selective pharmacologic inhibition in GCA monocytes., Results: GCA monocytes exhibited hallmark molecular features of TI. Specifically, these included enhanced IL-6 production upon stimulation, typical immunometabolic changes (e.g. increased glycolysis and glutaminolysis) and epigenetic changes promoting enhanced transcription of genes governing pro-inflammatory activation. Immunometabolic changes of TI (i.e. glycolysis) were a feature of myelomonocytic cells in GCA lesions and were required for enhanced cytokine production., Conclusions: Myelomonocytic cells in GCA activate TI programs sustaining enhanced inflammatory activation with excess cytokine production., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Analyzing gene expression profiles (GEP) through artificial intelligence provides meaningful insight into cancer disease. This study introduces DeepSHAP Autoencoder Filter for Genes Selection (DSAF-GS), a novel deep learning and explainable artificial intelligence-based approach for feature selection in genomics-scale data. DSAF-GS exploits the autoencoder's reconstruction capabilities without changing the original feature space, enhancing the interpretation of the results. Explainable artificial intelligence is then used to select the informative genes for chronic lymphocytic leukemia prognosis of 217 cases from a GEP database comprising roughly 20,000 genes. The model for prognosis prediction achieved an accuracy of 86.4%, a sensitivity of 85.0%, and a specificity of 87.5%. According to the proposed approach, predictions were strongly influenced by CEACAM19 and PIGP, moderately influenced by MKL1 and GNE, and poorly influenced by other genes. The 10 most influential genes were selected for further analysis. Among them, FADD, FIBP, FIBP, GNE, IGF1R, MKL1, PIGP, and SLC39A6 were identified in the Reactome pathway database as involved in signal transduction, transcription, protein metabolism, immune system, cell cycle, and apoptosis. Moreover, according to the network model of the 3D protein-protein interaction (PPI) explored using the NetworkAnalyst tool, FADD, FIBP, IGF1R, QTRT1, GNE, SLC39A6, and MKL1 appear coupled into a complex network. Finally, all 10 selected genes showed a predictive power on time to first treatment (TTFT) in univariate analyses on a basic prognostic model including IGHV mutational status, del(11q) and del(17p), NOTCH1 mutations, β2-microglobulin, Rai stage, and B-lymphocytosis known to predict TTFT in CLL. However, only IGF1R [hazard ratio (HR) 1.41, 95% CI 1.08-1.84, P=0.013), COL28A1 (HR 0.32, 95% CI 0.10-0.97, P=0.045), and QTRT1 (HR 7.73, 95% CI 2.48-24.04, P<0.001) genes were significantly associated with TTFT in multivariable analyses when combined with the prognostic factors of the basic model, ultimately increasing the Harrell's c-index and the explained variation to 78.6% (versus 76.5% of the basic prognostic model) and 52.6% (versus 42.2% of the basic prognostic model), respectively. Also, the goodness of model fit was enhanced (χ2 = 20.1, P=0.002), indicating its improved performance above the basic prognostic model. In conclusion, DSAF-GS identified a group of significant genes for CLL prognosis, suggesting future directions for bio-molecular research., 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 Morabito, Adornetto, Monti, Amaro, Reggiani, Colombo, Rodriguez-Aldana, Tripepi, D’Arrigo, Vener, Torricelli, Rossi, Neri, Ferrarini, Cutrona, Gentile and Greco.)

Background and Purpose: Data are reported from the Italian CMT Registry., Methods: The Italian CMT Registry is a dual registry where the patient registers and chooses a reference center where the attending clinician collects a minimal dataset of information and administers the Charcot-Marie-Tooth (CMT) Examination/Neuropathy Score. Entered data are encrypted., Results: Overall, 1012 patients had registered (535 females) and 711 had received a genetic diagnosis. Demyelinating CMT (65.3%) was more common than axonal CMT2 (24.6%) and intermediate CMT (9.0%). The PMP22 duplication was the most frequent mutation (45.2%), followed by variants in GJB1 and MPZ (both ~10%) and MFN2 (3.3%) genes. A relatively high mutation rate in some "rare" genes (HSPB1 1.6%, NEFL 1.5%, SH3TC2 1.5%) and the presence of multiple mutation clusters across Italy was observed. CMT4A was the most disabling type, followed by CMT4C and CMT1E. Disease progression rate differed depending on the CMT subtype. Foot deformities and walking difficulties were the main features. Shoe inserts and orthotic aids were used by almost one-half of all patients. Scoliosis was present in 20% of patients, especially in CMT4C. Recessive forms had more frequently walking delay, walking support need and wheelchair use. Hip dysplasia occurred in early-onset CMT., Conclusions: The Italian CMT Registry has proven to be a powerful data source to collect information about epidemiology and genetic distribution, clinical features and disease progression of CMT in Italy and is a useful tool for recruiting patients in forthcoming clinical trials., (© 2023 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

By pairing to messenger RNAs (mRNAs for short), microRNAs (miRNAs) regulate gene expression in animals and plants. Accurately identifying which mRNAs interact with a given miRNA and the precise location of the interaction sites is crucial to reaching a more complete view of the regulatory network of an organism. Only a few experimental approaches, however, allow the identification of both within a single experiment. Computational predictions of miRNA-mRNA interactions thus remain generally the first step used, despite their drawback of a high rate of false-positive predictions. The major computational approaches available rely on a diversity of features, among which anchoring the miRNA seed and measuring mRNA accessibility are the key ones, with the first being universally used, while the use of the second remains controversial. Revisiting the importance of each is the aim of this paper, which uses Cross-Linking, Ligation, And Sequencing of Hybrids (CLASH) datasets to achieve this goal. Contrary to what might be expected, the results are more ambiguous regarding the use of the seed match as a feature, while accessibility appears to be a feature worth considering, indicating that, at least under some conditions, it may favour anchoring by miRNAs.

Liquid biopsy has dramatically changed cancer management in the last decade; however, despite the huge number of miRNA signatures available for diagnostic or prognostic purposes, it is still unclear if dysregulated miRNAs in the bloodstream could be used to develop miRNA-based therapeutic approaches. In one author's previous work, nine miRNAs were found to be dysregulated in early-stage colon cancer (CRC) patients by NGS analysis followed by RT-dd-PCR validation. In the present study, the biological effects of the targeting of the most relevant dysregulated miRNAs with anti-miRNA peptide nucleic acids (PNAs) were verified, and their anticancer activity in terms of apoptosis induction was evaluated. Our data demonstrate that targeting bloodstream up-regulated miRNAs using anti-miRNA PNAs leads to the down-regulation of target miRNAs associated with inhibition of the activation of the pro-apoptotic pathway in CRC cellular models. Moreover, very high percentages of apoptotic cells were found when the anti-miRNA PNAs were associated with other pro-apoptotic agents, such as sulforaphane (SFN). The presented data sustain the idea that the targeting of miRNAs up-regulated in the bloodstream with a known role in tumor pathology might be a tool for the design of protocols for anti-tumor therapy based on miRNA-targeting molecules.

Significant skeletal alterations are present in Chronic Lymphocytic Leukemia (CLL) patients; bone erosion, particularly evident in the long bone shaft, appeared increased in the progressive disease stage. Moreover, the partial colonization of the bone with reactive bone marrow we documented via PET-FDG imaging suggests that neoplastic cell overgrowth contributes to bone derangement. Indeed, cytokines released by leukemic B cells impair osteoblast differentiation and enhance osteoclast formation in vitro. CD16, Fcγ-RIIIa, has been previously indicated as a marker of osteoclast precursors. We demonstrate, here, that the percentage of circulating monocytes, CD16+, is significantly higher in CLL patients than in normal controls and directly correlated with the extent of bone erosion. When we assessed if healthy monocytes, treated with a CLL-conditioned medium, modulated RANK, RANKL and CD16, we observed that all these molecules were up-regulated and CD16 to a greater extent. Altogether, these findings suggest that leukemic cells facilitate osteoclast differentiation. Interestingly, the evidence that monocytes, polarized toward the M2 phenotype, were characterized by high CD16 expression and showed a striking propensity to differentiate toward osteoclasts may provide further explanations for the enhanced levels of bone erosion detected, in agreement with the high number of immunosuppressive-M2 cells present in these patients.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures., (© 2022 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli., Competing Interests: M.C. and G.C. have been supported by Gilead. The other authors declare no conflict of interest.

Objective: We aim to show the step-by-step surgical technique of mitral valve re-repair by means of a repeated right anterior minithoracotomy in a case of a procedure-related early mitral valve repair failure due to left ventricular positive remodeling and chordal pseudo-elongation., Methods: The patient was readdressed to our institution for an early severe mitral valve regurgitation, less than a year after performing a right minithoracotomy mitral valve repair (42-mm annular ring implantation, P2 triangular resection, and P2 neochord positioning). The mechanism was attributed to a positive left ventricle remodeling and neochordal pseudo-elongation. Therefore, we decided to perform a mitral valve re-repair in a redo minimally invasive cardiac surgery. We describe in a video-guided step-by-step fashion the surgical procedure, from the reopening of the right anterior minithoracotomy to the surgical strategy chosen to address the re-repair, guided by the mechanism of the previous repair failure., Results: We replaced the previously implanted ring with a smaller one and positioned a new polytetrafluoroethylene 4-0 neochord at the P2 level. The patient was discharged home on the fifth postoperative day after an uneventful hospital stay. Predischarge echocardiogram demonstrated undetectable residual mitral valve regurgitation. At 3-month follow-up, echocardiographic and clinical data were encouraging. At 9-month follow-up, the patient endorsed no recurrence of cardiologic symptoms., Conclusions: Redo minimally invasive cardiac surgery is a viable option even in case of a mitral valve re-repair due to previous repair failure, especially when procedure related in degenerative mitral disease. Combining the benefits of mitral valve re-repair with those of a minimally invasive surgery may optimize short-term and long-term outcomes.

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rβ1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rβ1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rβ1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rβ1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rβ1-positive CLL cells in the spleen. Our findings indicate that IL-12Rβ1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In this protocol, we describe how to generate 3D culture surrogates of chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) bone marrow microenvironments. We detail the use of culturing scaffolds populated with BM stromal cells and tumor cells in the RCCS™ bioreactor. This 3D culture can efficiently recapitulate tumor-stroma crosstalk and allows the testing of drugs such as ibrutinib and bortezomib. Moreover, this protocol can be used for the generation of other and more complex tumor microenvironments. For complete details on the use and execution of this protocol, please refer to Belloni et al. (2018) and Barbaglio et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely to benefit from mAbs treatment is currently challenging and limiting the impact of such therapies. To overcome this issue, and to fulfill the expectations of mAbs therapies, it is urgently required to develop proper culture models capable of faithfully reproducing the interactions between tumor and its surrounding native microenvironment (TME). Three-dimensional (3D) models which allow the assessment of the impact of drugs on tumors within its TME in a patient-specific context are promising avenues to progressively fill the gap between conventional 2D cultures and animal models, substantially contributing to the achievement of personalized medicine. This review aims to give a brief overview of the currently available 3D models, together with their specific exploitation for therapeutic mAbs testing, underlying advantages and current limitations to a broader use in preclinical oncology.

Viruses have evolved numerous mechanisms to exploit the molecular machinery of their host cells, including the broad spectrum of host RNA-binding proteins (RBPs). However, the RBP interactomes of most viruses are largely unknown. To shed light on the interaction landscape of RNA viruses with human host cell RBPs, we have analysed 197 single-stranded RNA (ssRNA) viral genome sequences and found that the majority of ssRNA virus genomes are significantly enriched or depleted in motifs for specific human RBPs, suggesting selection pressure on these interactions. To facilitate tailored investigations and the analysis of genomes sequenced in future, we have released our methodology as a fast and user-friendly computational toolbox named SMEAGOL. Our resources will contribute to future studies of specific ssRNA virus-host cell interactions and support the identification of antiviral drug targets.

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5 + B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5 + B cells, nor in specific B-cell subpopulations or the CD5 + cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin., 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 © 2022 Bagnara, Colombo, Reverberi, Matis, Massara, Cardente, Ubezio, Agostini, Agnelli, Neri, Cardillo, Vergani, Ghiotto, Mazzarello, Morabito, Cutrona, Ferrarini and Fais.)

Methods allowing for in situ dosimetry and range verification are essential in radiotherapy to reduce the safety margins required to account for uncertainties introduced in the entire treatment workflow. This study suggests a non-invasive dosimetry concept for carbon ion radiotherapy based on phase-change ultrasound contrast agents. Injectable nanodroplets made of a metastable perfluorobutane (PFB) liquid core, stabilized with a crosslinked poly(vinylalcohol) shell, are vaporized at physiological temperature when exposed to carbon ion radiation (C-ions), converting them into echogenic microbubbles. Nanodroplets, embedded in tissue-mimicking phantoms, are exposed at 37 °C to a 312 MeV/u clinical C-ions beam at different doses between 0.1 and 4 Gy. The evaluation of the contrast enhancement from ultrasound imaging of the phantoms, pre- and post-irradiation, reveals a significant radiation-triggered nanodroplets vaporization occurring at the C-ions Bragg peak with sub-millimeter shift reproducibility and dose dependency. The specific response of the nanodroplets to C-ions is further confirmed by varying the phantom position, the beam range, and by performing spread-out Bragg peak irradiation. The nanodroplets' response to C-ions is influenced by their concentration and is dose rate independent. These early findings show the ground-breaking potential of polymer-shelled PFB nanodroplets to enable in vivo carbon ion dosimetry and range verification., (© 2022. The Author(s).)

Considering the emergence of antifungal resistance on Sporothrix brasiliensis, we aimed to assess new benzylidene-carbonyl compounds against feline-borne S. brasiliensis isolates. The compounds were designed as bioisosteres from previously reported benzylidene-ketones generating the p-coumaric (1), cinnamic (2), p-methoxycinnamic (3) and caffeic acid (4) analogues. The corresponding compounds were tested against feline isolates of S. brasiliensis with sensitivity (n = 4) and resistance (n = 5) to itraconazole (ITZ), following the M38-A2 protocol (CLSI, Reference method for broth dilution antifungal susceptibility testing of filamentous fungi M38-A2 Guideline, 2008). Eleven analogues showed activity against all fungal strains with minimum inhibitory concentrations (MIC) ≤1 mg/ml (1a-d, 2e, 3b, 3e, 4, 4a and 5e) and fungicidal concentrations (MFC) ≤1 mg/ml (1b, 1d, 3e and 4a), whereas 3 was the less active with both MIC and MFC values above 1 mg/ml. Compound 3e (4-methoxy-N-butylcinnamamide) was the most potent (MIC range 0.08-0.16 mg/ml; MFC range 0.32-0.64 mg/ml) from the set, suggesting a different role of the substituents in ester and amide derivatives. The designed compounds proved to be important prototypes with improved drug-likeness to achieve compounds with higher activity against ITZ-resistant S. brasiliensis., (© 2021 John Wiley & Sons A/S.)

Motivation: The increasing availability of metabolomic data and their analysis are improving the understanding of cellular mechanisms and how biological systems respond to different perturbations. Currently, there is a need for novel computational methods that facilitate the analysis and integration of increasing volume of available data. Results: In this paper, we present Totoro a new constraint-based approach that integrates quantitative non-targeted metabolomic data of two different metabolic states into genome-wide metabolic models and predicts reactions that were most likely active during the transient state. We applied Totoro to real data of three different growth experiments (pulses of glucose, pyruvate, succinate) from Escherichia coli and we were able to predict known active pathways and gather new insights on the different metabolisms related to each substrate. We used both the E. coli core and the iJO1366 models to demonstrate that our approach is applicable to both smaller and larger networks. Availability: Totoro is an open source method (available at https://gitlab.inria.fr/erable/totoro) suitable for any organism with an available metabolic model. It is implemented in C++ and depends on IBM CPLEX which is freely available for academic purposes., Competing Interests: Author AJ-L was employed by Soladis GmBH. The remaining 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 © 2022 Galvão Ferrarini, Ziska, Andrade, Julien-Laferrière, Duchemin, César, Mary, Vinga and Sagot.)

(1) Background: Charcot-Marie-Tooth disease (CMT) is the most frequent form of inherited chronic motor and sensory polyneuropathy. Over 100 CMT causative genes have been identified. Previous reports found PMP22 , GJB1 , MPZ , and MFN2 as the most frequently involved genes. Other genes, such as BSCL2 , MORC2 , HINT1 , LITAF , GARS , and autosomal dominant GDAP1 are responsible for only a minority of CMT cases. (2) Methods: we present here our records of CMT patients harboring a mutation in one of these rare genes ( BSCL2 , MORC2 , HINT1 , LITAF , GARS , autosomal dominant GDAP1 ). We studied 17 patients from 8 unrelated families. All subjects underwent neurologic evaluation and genetic testing by next-generation sequencing on an Ion Torrent PGM (Thermo Fischer) with a 44-gene custom panel. (3) Results: the following variants were found: BSCL2 c.263A > G p.Asn88Ser (eight subjects), MORC2 c.1503A > T p.Gln501His (one subject), HINT1 c.110G > C p.Arg37Pro (one subject), LITAF c.404C > G p.Pro135Arg (two subjects), GARS c.1660G > A p.Asp554Asn (three subjects), GDAP1 c.374G > A p.Arg125Gln (two subjects). (4) Expanding the spectrum of CMT phenotypes is of high relevance, especially for less common variants that have a higher risk of remaining undiagnosed. The necessity of reaching a genetic definition for most patients is great, potentially making them eligible for future experimentations.

Background: The rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions., Results: We sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino acids required for insect development and cuticle biosynthesis., Conclusions: Here we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes., (© 2021. The Author(s).)

Background: A biallelic intronic AAGGG repeat expansion in the Replication Factor C subunit 1 (RFC1) gene has been recently associated with Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome, a disorder often presenting as a slowly evolving sensory neuropathy at the onset. "Chronic Idiopathic Axonal Polyneuropathy" (CIAP) is a common indolent axonal neuropathy of adulthood which remains without an identifiable cause despite thorough investigations., Methods: We screened 234 probands diagnosed with CIAP for a pathogenic biallelic RFC1 AAGGG repeat expansion. Patients were selected from 594 consecutive patients with neuropathy referred to our tertiary-care center for a sural nerve biopsy over 10 years., Results: The RFC1 AAGGG repeat expansion was common in patients with pure sensory neuropathy (21/40, 53%) and less frequent in cases with predominantly sensory (10/56, 18%, P < 0.001) or sensorimotor (3/138, 2%, P < 0.001) neuropathy. The mutation was associated with sensory ataxia (τ b  = 0.254, P < 0.001), autonomic disturbances (35% vs 8%, Prevalence Odds Ratio-POR 6.73 CI 95% 2.79-16.2, P < 0.001), retained deep tendon reflexes (score 18.0/24 vs 11.5/24, R = 0.275, P < 0.001). On pathology, we observed absent/scant regenerative changes (τ b  = - 0.362, P < 0.001), concomitant involvement of large (100% and 99%, n.s.), small myelinated (97% vs 81%, POR 7.74 CI 95% 1.03-58.4, P = 0.02) and unmyelinated nerve fibers (85% vs 41%, POR 8.52 CI 95% 3.17-22.9, P < 0.001). Cerebellar or vestibular involvement was similarly rare in the two groups., Conclusions: This study highlights the frequent occurrence of the RFC1 AAGGG repeat expansion in patients diagnosed with CIAP and characterizes the clinical and pathological features of the related neuro(no)pathy., (© 2021. The Author(s).)

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation., (© 2021 by The American Society of Hematology.)

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.