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The use of light detection and ranging technologies, i.e. terrestrial laser scanner (TLS), airborne laser scanner (ALS) and mobile laser scanner (MLS), together with the unmanned aerial vehicles digital photogrammetry (UAV-DP) and satellite data are proving to be fundamental tools to carry out reliable muographic measurement campaigns. The main purpose of this paper is to propose a workflow to correctly plan and exploit these types of data for muon radiography aims. To this end, a real case study is presented: searching for hidden tombs in the Etruscan necropolis of Palazzone (Umbria, Italy). A high-resolution digital elevation model (DEM) and three-dimensional models of the ground surface/sub-surface of the study area were created by merging data obtained using different survey methods to achieve the most accurate three-dimensional environment. Indeed, the simulated muon flux transmission used to infer relative transmission values, and the estimated density distribution, depends on the reliability of the three-dimensional reconstructed ground surface model. The aim of this study is to provide knowledge on the use of TLS and UAV-DP data and GPS-acquired points within the transmission-based muography process and how these data could improve or worsen the muon imaging results. Moreover, this study confirmed that muography applications require a multidisciplinary approach., (© 2023. The Author(s).)

Artificial light at night (ALAN) is a globally spreading anthropogenic stressor, affecting more than 20% of coastal habitats. The alteration of the natural light/darkness cycle is expected to impact the physiology of organisms by acting on the complex circuits termed as circadian rhythms. Our understanding of the impact of ALAN on marine organisms is lagging behind that of terrestrial ones, and effects on marine primary producers are almost unexplored. Here, we investigated the molecular and physiological response of the Mediterranean seagrass, Posidonia oceanica (L.) Delile, as model to evaluate the effect of ALAN on seagrass populations established in shallow waters, by taking advantage of a decreasing gradient of dim nocturnal light intensity (from < 0.01 to 4 lx) along the NW Mediterranean coastline. We first monitored the fluctuations of putative circadian-clock genes over a period of 24 h along the ALAN gradient. We then investigated whether key physiological processes, known to be synchronized with day length by the circadian rhythm, were also affected by ALAN. ALAN influenced the light signalling at dusk/night in P. oceanica, including that of shorter blue wavelengths, through the ELF3-LUX1-ZTL regulatory network, and suggested that the daily perturbation of internal clock orthologs in seagrass might have caused the recruitment of PoSEND33 and PoPSBS genes to mitigate the repercussions of a nocturnal stress on photosynthesis during the day. A long-lasting impairment of gene fluctuations in sites characterised by ALAN could explain the reduced growth of the seagrass leaves when these were transferred into controlled conditions and without lighting during the night. Our results highlight the potential contribution of ALAN to the global loss of seagrass meadows, posing questions about key interactions with a variety of other human-related stressors in urban areas, in order to develop more efficient strategies to globally preserve these coastal foundation species., (© 2023. The Author(s).)

Muon radiography (muography) is an imaging technique based on atmospheric muon absorption in matter that allows to obtain two and three-dimensional images of internal details of hidden objects or structures. The technique relies on atmospheric muon flux measurements performed around and underneath the object under examination. It is a non-invasive and passive technique and thus can be thought of as a valid alternative to common prospecting techniques used in archaeological, geological and civil security fields. This paper describes muon radiography measurements, in the context of archaeological and geological studies carried out at the Temperino mine (LI, Tuscany, Italy), for the search and three-dimensional visualisation of cavities. This mine has been exploited since Etruscan times until recently (1973), and is now an active tourist attraction with public access to the tunnels. Apart from the archaeological interest, the importance of mapping the cavities within this mine lies in identifying the areas where the extraction ores were found and also in the safety issues arising from the tourist presence inside the mine. The three-dimensional imaging is achieved with two different algorithms: one involving a triangulation of two or more measurements at different locations; the other, an innovative technique used here for the first time, is based on the back-projections of reconstructed muon tracks. The latter requires only a single muographic data tacking and is to be preferred in applications where more than one site location can be difficult to access. Finally the quality of the three-dimensional muographic imaging was evaluated by comparing the results with the laser scan profiles obtained for some known cavities within the Temperino mine., (© 2022. The Author(s).)

Bipolar affective disorder (BD) is a severe psychiatric illness, for which lithium (Li) is the gold standard for acute and maintenance therapies. The therapeutic response to Li in BD is heterogeneous and reliable biomarkers allowing patients stratification are still needed. A GWAS performed by the International Consortium on Lithium Genetics (ConLiGen) has recently identified genetic markers associated with treatment responses to Li in the human leukocyte antigens (HLA) region. To better understand the molecular mechanisms underlying this association, we have genetically imputed the classical alleles of the HLA region in the European patients of the ConLiGen cohort. We found our best signal for amino-acid variants belonging to the HLA-DRB1*11:01 classical allele, associated with a better response to Li (p < 1 × 10 -3 ; FDR < 0.09 in the recessive model). Alanine or Leucine at position 74 of the HLA-DRB1 heavy chain was associated with a good response while Arginine or Glutamic acid with a poor response. As these variants have been implicated in common inflammatory/autoimmune processes, our findings strongly suggest that HLA-mediated low inflammatory background may contribute to the efficient response to Li in BD patients, while an inflammatory status overriding Li anti-inflammatory properties would favor a weak response., (© 2021. The Author(s).)

Biofilm formation poses an important clinical trouble due to resistance to antimicrobial agents; therefore, there is an urgent demand for new antibiofilm strategies that focus on the use of alternative compounds also in combination with conventional drugs. Drug-tolerant persisters are present in Candida albicans biofilms and are detected following treatment with high doses of amphotericin B. In this study, persisters were found in biofilms treated with amphotericin B of two clinical isolate strains, and were capable to form a new biofilm in situ. We investigated the possibility of eradicating persister-derived biofilms from these two Candida albicans strains, using the peptide gH625 analogue (gH625-M). Confocal microscopy studies allowed us to characterize the persister-derived biofilm and understand the mechanism of interaction of gH625-M with the biofilm. These findings confirm that persisters may be responsible for Candida biofilm survival, and prove that gH625-M was very effective in eradicating persister-derived biofilms both alone and in combination with conventional antifungals, mainly strengthening the antibiofilm activity of fluconazole and 5-flucytosine. Our strategy advances our insights into the development of effective antibiofilm therapeutic approaches.

We have investigated the crossing of the blood brain barrier (BBB) by the peptide gH625 and compared to the uptake by liver in vivo. We clearly observed that in vivo administration of gH625 allows the crossing of the BBB, although part of the peptide is sequestered by the liver. Furthermore, we used a combination of biophysical techniques to gain insight into the mechanism of interaction with model membranes mimicking the BBB and the liver. We observed a stronger interaction for membranes mimicking the BBB where gH625 clearly undergoes a change in secondary structure, indicating the key role of the structural change in the uptake mechanism. We report model studies on liposomes which can be exploited for the optimization of delivery tools.

Candida parapsilosis is one of the most common causes of candidiasis, particularly in the very young and the very old. Studies of gene function are limited by the lack of a sexual cycle, the diploid genome, and a paucity of molecular tools. We describe here the development of a plasmid-based CRISPR-Cas9 system for gene editing in C. parapsilosis. A major advantage of the system is that it can be used in any genetic background, which we showed by editing genes in 20 different isolates. Gene editing is carried out in a single transformation step. The CAS9 gene is expressed only when the plasmid is present, and it can be removed easily from transformed strains. There is theoretically no limit to the number of genes that can be edited in any strain. Gene editing is increased by homology-directed repair in the presence of a repair template. Editing by non-homologous end joining (NHEJ) also occurs in some genetic backgrounds. Finally, we used the system to introduce unique tags at edited sites.

p.(Arg320His) mutation in the KCNC1 gene in human 11p15.1 has recently been identified in patients with progressive myoclonus epilepsies, a group of rare inherited disorders manifesting with action myoclonus, myoclonic epilepsy, and ataxia. This KCNC1 variant causes a dominant-negative effect. Here we describe three patients from the same family with intellectual disability and dysmorphic features. The three affected individuals carry a c.1015C>T (p.(Arg339*)) nonsense variant in KCNC1 gene. As previously observed in the mutant mouse carrying a disrupted KCNC1 gene, these findings reveal that individuals with a KCNC1 loss-of-function variant can present intellectual disability without seizure and epilepsy.

Antimicrobial peptides are promising candidates as future therapeutics in order to face the problem of antibiotic resistance caused by pathogenic bacteria. Myxinidin is a peptide derived from the hagfish mucus displaying activity against a broad range of bacteria. We have focused our studies on the physico-chemical characterization of the interaction of myxinidin and its mutant WMR, which contains a tryptophan residue at the N-terminus and four additional positive charges, with two model biological membranes (DOPE/DOPG 80/20 and DOPE/DOPG/CL 65/23/12), mimicking respectively Escherichia coli and Pseudomonas aeruginosa membrane bilayers. All our results have coherently shown that, although both myxinidin and WMR interact with the two membranes, their effect on membrane microstructure and stability are different. We further have shown that the presence of cardiolipin plays a key role in the WMR-membrane interaction. Particularly, WMR drastically perturbs the DOPE/DOPG/CL membrane stability inducing a segregation of anionic lipids. On the contrary, myxinidin is not able to significantly perturb the DOPE/DOPG/CL bilayer whereas interacts better with the DOPE/DOPG bilayer causing a significant perturbing effect of the lipid acyl chains. These findings are fully consistent with the reported greater antimicrobial activity of WMR against P. aeruginosa compared with myxinidin.

Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the alpha-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14).

The NFKB-2 (Lyt-10) gene codes for an NF-kappaB-related transcription factor containing rel-polyG-ankyrin domains. Rearrangements of the NFKB-2 locus leading to the production of 3' truncated NFKB-2 proteins are recurrently found in lymphoid neoplasms, particularly cutaneous lymphomas. Such mutant NFKB-2 proteins have lost the ability to repress transcription that is typical of NFKB-2 subunit p52, and function as constitutive transcriptional activators. To verify whether the expression of abnormal NFKB-2 proteins can lead to malignant transformations in mammalian cells, we transfected human lymphoblastoid cell lines and murine fibroblasts (Balb/3T3) with expression vectors carrying the cDNAs coding for normal NFKB-2p52, Lyt-10C alpha or LB40 proteins, which are representative of the abnormal types found in lymphoma cases. The expression of both normal and mutant NFKB-2 proteins has a lethal effect on lymphoblastoid cells and a cytotoxic effect was also observed in murine fibroblasts. The fibroblast cell lines expressing Lyt-10C alpha or LB40, but not those expressing normal NFKB-2p52, were capable of forming colonies in soft agar. The analysis of individual clones revealed that cloning efficiency correlated with the expression levels of the abnormal proteins. Injection of the Lyt-10C alpha-transfected Balb cells in SCID mice led to tumor formation in all of the animals, whereas no tumors were observed in the mice injected with control or NFKB-2p52-transfected cells, thus indicating that abnormal NFKB-2 protein expression is tumorigenic in vivo. Our results show that mutant NFKB-2 proteins can lead to the transformed phenotype, and support the hypothesis that alterations in NFKB-2 genes may play a role in lymphomagenesis.

Rearrangements of the NFKB2 gene are associated with lymphoid malignancies, but the functional significance of these alterations is not known. Here we characterize structurally and functionally a rearranged NFKB2 gene identified at the T cell lymphoma line, HUT78. The rearrangement has truncated NFKB2 sequences within the 3' ankyrin domain, leading to the production of truncated mRNA species and proteins as detected by Northern blot and immunoprecipitation analysis, respectively. Cloning and sequencing of the corresponding cDNAs indicates that, via alternative splicing, the rearranged gene codes for two proteins of 84 and 85 kD (p84/85) which retain the DNA-binding rel domain and the first five ankyrin repeats, but have lost their carboxy-terminus including the seventh ankyrin repeat. Immunofluorescence and immunoprecipitation analysis of HUT78 cells indicate that p84/85 are abnormally located in the nucleus in an unprocessed form, suggesting that these proteins can escape the cytoplasmic retention typical of the normal NFKB2 p100 protein before it is processed into p52. Electrophoretic mobility shift assays performed on HUT78 nuclear extracts indicate that the abnormal NFKB2 proteins bind kappa B sites specifically and alter the composition of NF-kappa B complexes in HUT78 cells. Transient co-transfection assays involving NFKB2 expression vectors and kappa B-driven reporter plasmids indicate that NFKB2 p85 has lost the transcriptional repressor functions typical of normal NFKB2 p52. These data indicate that the NFKB2 gene rearrangement detected in HUT78 cells leads to the production of abnormal NFKB2 proteins capable of altering the function of the NF-kappa B transcription system. Since analogous rearrangements are found in lymphoid malignancies, these findings further support a role of NFKB2 alterations in tumorigenesis.

The NFKB-2 gene (previously LYT-10, NF-kappa Bp100 or NF-kappa Bp97) codes for a NF-kappa B/rel related protein which is highly homologous to NFKB-1 (previously NF-kappa Bp105) within its rel, poly-glycine and ankyrin domains. The NFKB-2 gene is a candidate proto-oncogene since it is involved in lymphoma-associated chromosomal aberrations. In order to gain insight into the physiological function and role in tumorigenesis of NFKB-2, we have analysed its mechanism of expression and role in transcriptional regulation. We report that, contrary to previous studies, a single 3.2 kb mRNA species and its 100 kD (p100) primary translation product is detectable in all cell types tested. A second NFKB-2 protein, p52, corresponding to the amino-terminal half (rel domain) of NFKB-2 p100, is detectable in the same cell types and derives from the post-translational processing of p100. While p100 is constitutively localized in the cytoplasm, NF-kappa B induction by TPA treatment of Hela cells is associated with cytoplasmic/nuclear translocation of NFKB-2 p52 and its appearance within DNA-binding NF-kappa B complexes. NFKB-2 p52 differs from NFKB-1p50 in its differential affinity for kappa B sequences: by itself it binds H2/HLA-kappa B sites more efficiently than HIV/IgK-kappa B sites, while it can bind both sites efficiently when complexed with Rel-A(p65). Transient co-transfection of expression and reporter plasmids in cells devoid of endogenous NF-kappa B activity showed that p52 has no intrinsic transcriptional activation capabilities: it can stimulate Rel-A(p65)-driven transcription by formation of p65/p52 heterodimers, whereas, overexpressed, it down-regulates p65-dependent transcription by formation of inactive p52/p52 homodimers. These results indicate that the NFKB-2 gene codes for an inducible NF-kappa B transcription factor with the capability of differentially regulating NF-kappa B transcription depending on its abundance in the nucleus.

We have previously reported the identification of a novel putative proto-oncogene involved in the breakpoint of a t(10;14)(q24;q32) chromosomal translocation in a case of B-cell lymphoma. This gene, called lyt-10 (NFKB2/p52), is a member of the NF-kappa B family of transcription factors and displays a high degree of homology with the NFKB1/p50. Here we describe the genomic organization of the lyt-10 gene based on the restriction analysis of genomic phage clones and the sequence determination of exon-intron boundaries. The lyt-10 gene spans a genomic region of about 8 kb on 10q24, and contains 24 exons, ranging in size between 41 and 258 base pairs. To improve the understanding of the role of lyt-10 in lymphomagenesis, we performed Southern blot analysis to detect alterations of the lyt-10 gene in a large panel of cases representative of different types of lymphoid malignancies. We found rearrangements in 5 of 228 (approximately 2%) cases analysed: two cases of B-cell lymphoma, one case of multiple myeloma and two cases of T-cell lymphoma. The use of various probes specific for different regions of the lyt-10 locus revealed that rearrangements in positive cases lead to the partial or total deletion of the carboxy-terminal region containing the ankyrin domain. Taken together, our results indicate that lyt-10 gene rearrangements represent a recurrent lesion that may be involved in the pathogenesis of both B- and T-cell malignancies, and suggest that truncation of the ankyrin domain may be a common mechanism of lesion leading to abnormal lyt-10 activation in lymphoid neoplasia.

Negative feedback regulation of c-myc gene expression has been observed in some, but not all, cell types. In order to demonstrate conclusively the existence of this mechanism and gain insight into the cause of its inactivation, we have directly examined its function in B cells and then investigated its activity in a number of cell types. We demonstrate the existence of negative c-myc autoregulation by showing the rapid, dose dependent and reversible suppression of endogenous c-myc expression in EBV-immortalized B lymphoblastoid cells transfected with a c-myc gene expressed under the control of a heavy metal inducible promoter. Autoregulation occurs at the level of transcriptional initiation and is mediated by at least one stable intermediate or cofactor molecule. The c-myc autoregulatory mechanism was found operative in all (11 of 11) non-tumorigenic cells tested, including normal and immortalized lymphocytes and fibroblasts. However, this mechanism was found to be inactive in all (10 of 10) tumor cell lines derived from a variety of tissues including those carrying normal and oncogenically activated c-myc genes. These data establish the existence of an important regulatory circuit modulating c-myc expression in normal cells and suggest that its inactivation may represent a general regulatory disturbance of transformed cells.