Confirmatory validation analysis of the PROFFIT questionnaire to assess financial toxicity in cancer patients.

BACKGROUND: The Patient Reported Outcome for Fighting FInancial Toxicity (PROFFIT) questionnaire was developed to measure financial toxicity (FT) and identify its determinants. The aim of the present study was to confirm its validity in a prospective cohort of patients receiving anticancer treatment. PATIENTS AND METHODS: From March 2021 to July 2022, 221 patients were enrolled at 10 Italian centres. Selected items of the EORTC-QLQ-C30 questionnaire represented the anchors, specifically, question 28 (Q-28) on financial difficulties, and questions 29-30 measuring global health status/quality of life (HR-QOL). The study had 80% power to detect a 0.20 correlation coefficient (r) between anchors and PROFFIT-score (items 1-7, range 0-100, 100 indicating maximum FT) with bilateral alpha 0.05 and 80% power. Confirmatory factor analysis was conducted. FT determinants (items 8-16) were described. RESULTS: Median age of patients was 65 years, 116 (52.5%) were females, 96 (43.4%) had low education level. Confirmatory factor analysis confirmed goodness of fit of the PROFFIT-score. Significant partial correlation of PROFFIT-score was found with Q-28 (r = 0.51) and HR-QOL (r = -0.23). Mean (SD) PROFFIT-score at baseline was 36.5 (24.9); it was statistically significantly higher for patients living in South Italy, those with lower education level, those who were freelancer/unemployed at diagnosis and those who reported significant economic impact from the COVID-19 pandemic. Mean (SD) scores of determinants ranged from 17.6 (27.1) for item 14 (support from medical staff) to 49.0 (36.3) for item 10 (expenses for medicines or supplements). PROFFIT-score significantly increased with worsening response to determinants. CONCLUSIONS: External validation of PROFFIT-score in an independent sample of patients was successful. The instrument is now being used in clinical studies.

Tuning the biomimetic performances of 4-hydroxyproline-containing cyclic peptoids.

Five new cyclic peptoids containing (2S,4R)-4-hydroxyproline (Hyp) residues have been designed and synthesized using a mixed "submonomer/monomer" approach. Alkali metal cation affinities and ion transport activities were assessed by experimental (NMR and HPTS assay in liposomes) and computational methods. Easy functionalization of hydroxyproline residues afforded a bouquet of cyclic oligomers showing correlation between ion transport abilities and cytotoxic activities on selected human cancer cell lines.

Correction to: Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming.

Unfortunately, the second author name was wrongly published in the original publication. The correct author name should read as follows.

Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming.

KEY MESSAGE: Transcriptional activation of genes belonging to the plastidial MEP-derived isoprenoid pathway by elicitation with methyl jasmonate and coronatine enhanced the content of bioactive abietane diterpenes in Salvia sclarea hairy roots. We have shown that aethiopinone, an abietane diterpene synthesized in Salvia sclarea roots is cytotoxic and induces apoptosis in human melanoma cells. To develop a production platform for this compound and other abietane diterpenes, hairy root technology was combined with the elicitation of methyl jasmonate (MeJA) or the phytotoxin coronatine (Cor). Both MeJA and Cor induced a significant accumulation of aethiopinone, but prolonged exposure to MeJA irremediably caused inhibition of hairy root growth, which was unaffected by Cor treatment. Considering together the fold increase in aethiopinone content and the final hairy root biomass, the best combination was a Cor treatment for 28 days, which allowed to obtain up to 105.34 ± 2.30 mg L-1 of this compound to be obtained, corresponding to a 24-fold increase above the basal content in untreated hairy roots. MeJA or Cor elicitation also enhanced the synthesis of other bioactive abietane-quinone diterpenes. The elicitor-dependent steering effect was due to a coordinated transcriptional activation of several biosynthetic genes belonging to the plastidial MEP-derived isoprenoid pathway. High correlations between aethiopinone content and MeJA or Cor-elicited level of gene transcripts were found for DXS2 (r 2 = 0.99), DXR (r 2 = 0.99), and GGPPS (r 2 = 0.98), encoding enzymes acting upstream of GGPP, the common precursor of diterpenes and other plastidial-derived terpenes, as well as CPPS (r 2 = 0.99), encoding the enzyme involved in the first cyclization steps leading to copalyl-diphosphate, the precursor of abietane-like diterpenes. These results point to these genes as possible targets of metabolic engineering approaches to establish a more efficient production platform for such promising anti-proliferative plant-derived compounds.

Correction: Identification of the key structural elements of a dihydropyrimidinone core driving toward more potent Hsp90 C-terminal inhibitors.

Correction for 'Identification of the key structural elements of a dihydropyrimidinone core driving toward more potent Hsp90 C-terminal inhibitors' by S. Teracciano et al., Chem. Commun., 2016, 52, 12857-12860.

Identification of the key structural elements of a dihydropyrimidinone core driving toward more potent Hsp90 C-terminal inhibitors.

Hsp90 C-terminal modulation represents an attractive strategy for the development of potent and safer antitumor compounds. Continuing our investigation on DHPM type inhibitors here we report a new set of potent C-terminal ligands which allowed us to identify the key structural features crucial for the biological activity.

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes.

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I-II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II-III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

Genotoxic effects of the fungicide thiophanate-methyl on Podarcis sicula assessed by micronucleus test, comet assay and chromosome analysis.

The increasing use of pesticides in modern agriculture has raised the need to evaluate their potential threat to animal and human health. In the present study, the genotoxic effects of environmentally relevant exposure to the fungicide thiophanate-methyl (TM) were assessed in the lizard Podarcis sicula (Reptilia, Lacertidae) using micronucleus test, chromosome aberration analysis and single-cell gel electrophoresis (comet) assay. The number of micronuclei increased significantly with exposure time in lizard specimens exposed to 1.5% TM for 30-40 days. In situ hybridization with the specific HindIII centromeric satellite was positive in 18.7% of the micronuclei observed, suggesting an aneugenic effect of TM during mitosis. DNA damage, evaluated by the comet assay, documented a significant gain in comet length in relation to exposure time that was paralleled by a reduction in head size. Finally, cytogenetic analysis showed a significant increase in chromosome aberrations in exposed animals compared with controls. Our data suggest that long-term TM exposure induces a genomic damage that is positively correlated to exposure time. If such genotoxic effects arise so clearly in an ectothermal vertebrate, such as P. sicula, prolonged exposure TM must be considered as a cytogenetic hazard.

Lipovitellin constitutes the protein backbone of glycoproteins involved in sperm-egg interaction in the amphibian Discoglossus pictus.

Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120-kDa band released following gel-into-gel SDS-PAGE of both glycoproteins share the same N-terminal amino acid sequence, which itself is similar to the N-termini of Xenopus liver-synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120-kDa band is part of both gps 200 and 270/260. A 117-kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC-MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120-kDa band and is responsible for the formation of the 200-270-kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti-vitellogenin antibodies label only the surface of the dimple.

A transient asymmetric distribution of XNOA 36 mRNA and the associated spectrin network bisects Xenopus laevis stage I oocytes along the future A/V axis.

In Xenopus oogenesis, the mechanisms governing the localisation of molecules crucial for primary axis determination have been uncovered in recent years. In stage I oocytes, the mitochondrial cloud (MC) entraps RNAs implicated in germ line specification and other RNAs, such as Xwnt-11 and Xlsirts, that are later delivered to the vegetal pole. Microfilaments and microtubules gradually develop in the cytoplasm, sustaining organelles as well as the MC. At stage III, other mRNAs migrate to the vegetal hemisphere through a microtubule-dependent mechanism. We report here the isolation of a cDNA encoding XNOA 36, a highly conserved protein, whose function is to date not fully understood. The XNOA 36 transcript is abundantly accumulated in stage I oocytes where it decorates a filamentous network. At the end of stage I the transcript gradually segregates in a sector of the oocyte surrounding the MC and opposite the ovarian hylum. Here, XNOA 36 mRNA distributes in a gradient-like pattern extending from a peripheral network towards the interior of the oocyte. This distribution is similar to that of alpha-spectrin mRNA. Both mRNAs are segregated in one half of the 250 microm oocytes, with the MC located between the XNOA 36/alpha-spectrin mRNA-labelled and unlabelled regions. XNOA 36 mRNA localisation was uncoupled from that of alpha-spectrin mRNA by cytochalasin B or ice-nocodazole treatments, suggesting that the two transcripts rely on different mechanisms for their localisation. However, immunolocalisation experiments coupled with in situ hybridisation revealed that the XNOA 36 transcript co-localises with the protein spectrin. This observation, together with the finding that XNOA 36 mRNA co-precipitates with spectrin, indicates that these two molecules interact physically. In conclusion, our data suggest that XNOA 36 mRNA is localized and/or anchored in the oocyte through a cytoskeletal network containing spectrin. The putative implications of this finding are discussed.

Differential DMRT1 expression in the gonads of Podarcis sicula (Reptilia: Lacertidae).

DMRT genes encode a large family of transcription factors which share an unusual cysteine-rich DNA-binding motif, the DM domain. DM family members have been studied in the context of sexual development; in particular, the DMRT1 gene appeared to be the one most directly involved in sex determination, but its activity is largely unexplored and possible downstream targets of this factor have yet to be identified. DMRT1 of the lacertid lizard Podarcis sicula (PsDMRT1) was isolated as a model to study differential gene expression during the seasonal reproductive cycle of an ectothermal species. The adult testis of P. sicula exhibits full activity in spring, complete regression in summer and a slow autumnal recrudescence without spermiation. We cloned a 591-bp partial ORF of the PsDMRT1 fragment, whose putative amino acid sequence contains the conserved DM domain. Northern blot analysis of mRNA from different tissues of P. sicula individuals captured in spring demonstrated DMRT1 transcripts only in testis. Semi-quantitative RT-PCR and in situ hybridization experiments showed peak PsDMRT1 expression in spring, lower expression in autumn and no expression during the period of gonad regression. A possible correlation between androgen level variations and PsDMRT1 transcripts is hypothesized and discussed. Finally, data showed that PsDMRT1 is expressed only in spermatogenic cells, before the second meiotic division, suggesting that its role is confined to the proliferation and maintenance of spermatogonia and spermatocytes.

Phosphorylation of p27(BBP)/eIF6 and its association with the cytoskeleton are developmentally regulated in Xenopus oogenesis.

p27BBP/eIF6 is an evolutionarily conserved regulator of ribosomal function. It is necessary for 60S biogenesis and impedes improper joining of 40S and 60S subunits, regulated by protein kinase C or Efl1p. No data on p27BBP/eIF6 during early development of Metazoa are available. We studied the distribution, post-translational changes and association with the cytoskeleton of p27BBP/ eIF6 during Xenopus oogenesis and early development. Results indicate that p27BBP/eIF6 is present throughout oogenesis, partly associated with 60S subunits, partly free and with little cytoskeleton bound. During prophase I, p27BBP/eIF6 is detected as a single band of 27-kDa. Upon maturation induced by progesterone or protein kinase C, a serine-phosphorylated 29 kDa isoform appears and is kept throughout development to the neurula stage. Confocal microscopy showed that the distribution of p27BBP/eIF6 and its association with the cytoskeleton varies according to oogenesis stages. Briefly, in stage 6 oocytes, p27BBP/eIF6 has a limited dot-like distribution, and does not co-localize with cytokeratin, whereas upon maturation it spreads throughout the cytoplasm. After fertilization, a large fraction coalesces around cytomembranes and a cytochalasin B-sensitive co-localization with cytokeratin occurs. RNAse removes p27BBP/eIF6 from the cytokeratin fibres. Developmental data suggest a role of p27BBP/eIF6 in controlling ribosomal availability or regulating cross-talk between ribosomes and the cytoskeleton.

Primary structure and developmental expression of Dp ZP2, a vitelline envelope glycoprotein homolog of mouse ZP2, in Discoglossus pictus, one of the oldest living Anuran species.

A glycoprotein of the Xenopus vitelline envelope, gp 69/64, which mediates sperm binding, is closely related to the components of ZPA family, such as the mouse zona pellucida ZP2. To test the generality of these findings, we studied Discoglossus pictus, a species evolutionary distant from Xenopus and identified as a protein of 63 kDa in the vitelline envelope. Preliminary studies suggest that this protein may bind sperm at fertilization. We found that the 63-kDa protein is glycosylated and contains both N- and O-linked chains. We have cloned the cDNA encoding the Discoglossus protein of 63 kDa (Dp ZP2) by screening a Discoglossus cDNA library using Xenopus gp 69/64 cDNA as a probe. Analysis of the deduced sequence of Discoglossus protein revealed 48% identity with Xenopus gp 69/64 and 37-40% identity with mouse ZP2. The sequence conservation included a ZP domain, a potential furin cleavage site and a putative transmembrane domain. The N-terminus region of Dp ZP2 was 40% identical to the corresponding region of Xenopus gp 69/64 which has been shown to be essential for sperm binding to the VE. Although, as of yet, there is no evidence for sperm binding at the Dp ZP2 N-terminus, it is interesting that in this region three potential O-glycosylation sites are conserved in both species, in contrast to N-glycosylation sites. It was found that the Dp ZP2 mRNA is expressed in stage 1 oocytes and in the follicle cells surrounding the oocyte. Similarly, in Xenopus oocytes, the gp 69/64m RNA, was found in the oocytes, as well as in the somatic cells. Mol. Reprod. Dev. 59:133-143, 2001.

Following passage through the oviduct, the coelomic envelope of Discoglossus pictus (amphibia) acquires fertilizability upon reorganization, conversion of gp 42 to gp 40, extensive glycosylation, and formation of a specific layer.

This paper describes the morphological and biochemical changes in Discoglossus pictus coelomic oocyte envelope (CE) following passage through the oviduct. As in other anurans, in this species, the transformation of the envelope into vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein. In addition, several features, typical of Discoglossus pictus, were observed. A new layer, VE-D, forms underneath the VE region facing the site of sperm entrance, the dimple. In the VE, arrowhead-like bundles of fibrils are perpendicularly oriented toward the dimple. Ultrastructural observations and staining with UEA-I suggested that VE-D might have a role in supporting sperm penetration into the dimple by orienting VE bundles and exposing sugar residues such as fucose. In 'in vitro' tests, VE binding of sperm occurs only if sperm are exposed to A23187, in agreement with previous data (Campanella et al., 1997: Mol Reprod Dev 47:323-333). Sperm binding occurs all over the VE. Accordingly, extracts of the VE covering the animal or the vegetal hemisphere have the same affinity to lectins (DBA, DSA, GNA, MAA, SBA, SNA, UEA-I, WGA). The CE contains six main glycoproteins. Peptide mapping indicated that during CE transformation into VE, gp 42 shifts to an apparent M(r) of 40 and gp 61 is converted to an apparent M(r) of 63 kDa. Lectin blot analyses showed extensive changes in cross-reactivity of most glycoproteins during the CE-->VE transition. The fact that DBA and UEA-I stain gp 63 rather than gp 61 and that this change is related only to gp 63, suggested that O-glycosylation and terminal fucose might be acquired by gp 63 in preparation of fertilization. Gp 63 has recently been cloned (Vaccaro et al., submitted) and shown to exhibit high homology to Xenopus gp 69/64, a VE sperm ligand (Tian et al., 1997a: J. Cell Biol. 136: 1099-1108; Tian et al., 1997b: Dev Biol 187:143-153), and to ZP2 of mammals.

Enzyme activity in anuran spermatozoa upon induction of the acrosome reaction.

At the time of sperm-egg fusion in Discoglossus pictus, a large amount of electron-dense material of an unknown nature is liberated from the sperm. In the present work we studied this material in D. pictus sperm, using an assay utilising strips of autoradiographic film as a gelatin substrate for proteolytic enzymes. Upon treatment with A23187, D. pictus sperm produced a large halo on the gelatin substrate, indicating the presence of enzymes released by the sperm at the time of the acrosome reaction. In contrast, Xenopus laevis sperm did not produce halos upon treatment with A23187. The use of protease inhibitors such as TLCK, leupeptin, chymostatin, SBTI and EACA strongly suggests that the D. pictus whole acrosome contains trypsin and chymotrypsin activity while an SBTI-sensitive activity is absent in a small portion of the acrosome, possibly the anteriormost region. Furthermore, the material released at the acrosome reaction also contains an EACA-inhibited activity, indicating the presence of plasminogen activator. We conclude that D. pictus sperm release proteolytic enzyme(s) that may act at the egg surface at the time of gamete fusion.

alpha-Spectrin has a stage-specific asymmetrical localization during Xenopus oogenesis.

Xenopus oocyte organization largely depends upon the cytoskeleton distribution, which is dynamically regulated during oogenesis. An actin-based cytoskeleton is present in the cortex starting from stage 1. At stages 4-6, a complex and polarized cytoskeleton network forms in the cytoplasm. In this paper, we studied the distribution of spectrin, a molecule that has binding sites for several cytoskeletal proteins and is responsible for the determination of regionalized membrane territories. The localization of alpha-spectrin mRNA was analyzed during Xenopus oogenesis by in situ hybridization on both whole mount and sections, utilizing a cDNA probe encoding a portion of Xenopus alpha-spectrin. Furthermore, an antibody against mammalian alpha-spectrin was used to localize the protein. Our results showed a stage-dependent mRNA localization and suggested that spectrin may participate in the formation of specific domains in oocytes at stages 1 and 2 and 4-6. Mol. Reprod. Dev. 55:229-239, 2000.