Probing the interaction interface of the GADD45β/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry

GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103–117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113–136 and 259–274. Accordingly, an MKK7_KD Δ(101–136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues

Baseline physical functioning status of metastatic colorectal cancer patients predicts the overall survival but not the activity of a front-line oxaliplatin-fluoropyrimidine doublet

No differences in response rate (RR), progression-free survival (PFS), overall survival (OS) and quality of life (QoL) were seen in patients randomly treated with biweekly oxaliplatin plus either fluorouracil/folinic acid or capecitabine

Rationale and design of MILES-3 and MILES-4 studies: two randomized phase III trials comparing single-agent chemotherapy versus cisplatin-based doublets in elderly patients with advanced non-small cell lung cancer

BACKGROUND: Platinum-based chemotherapy is the cornerstone of treatment of advanced non-small-cell lung cancer (NSCLC) patients, but the efficacy of adding cisplatin to single-agent chemotherapy remains to be demonstrated in prospective phase III trials dedicated to elderly patients. Furthermore, the superiority of cisplatin/pemetrexed over cisplatin/gemcitabine in non-squamous NSCLC has not been confirmed prospectively. We present the rationale and design of two open-label, multicenter, randomized phase III trials for elderly patients with advanced NSCLC∶ Multicenter Italian Lung cancer in the Elderly Study (MILES)-3 and MILES-4. The aim is to evaluate the efficacy of adding cisplatin to single-agent chemotherapy (both trials) and the efficacy of pemetrexed versus gemcitabine in non-squamous tumors (MILES-4). PATIENTS AND METHODS: Both trials are dedicated to first-line therapy of patients older than 70 years with advanced NSCLC, ECOG performance status 0-1. In the MILES-3 trial, patients are randomized in a 1∶1 ratio to gemcitabine or cisplatin/gemcitabine. In the MILES-4 study patients with non-squamous histology are randomized, in a factorial design with 1∶1∶1∶1 ratio, to four arms: gemcitabine (A), cisplatin/gemcitabine (B), pemetrexed (C), cisplatin/pemetrexed (D). Two comparisons are planned∶ A+C vs B+D to test the role of cisplatin; A+B vs C+D to test the role of pemetrexed. Primary endpoint of both trials is overall survival. Secondary and exploratory endpoints include progression-free survival, response rate, toxicity, and quality of life. CONCLUSIONS: MILES-3 and MILES-4 results will add important evidence about the role of cisplatin-based doublets and pemetrexed in the first-line therapy of elderly patients with advanced NSCLC

Preclinical toxicology and safety pharmacology of the first-in-class GADD45β/MKK7 inhibitor and clinical candidate, DTP3

Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)β/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)β/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers

Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells

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Le TIC a supporto dell\u2019inclusione nella scuola dell\u2019infanzia: un passo non ancora futuro.

L'esperienza del periodo di chiusura delle scuole a causa della pandemia ha dimostrato quanto possa essere prezioso il supporto digitale nella didattica, ma anche di quanto l'interazione tra docente e discenti sia fondamentale per lo sviluppo socio-cognitivo degli alunni. L'interazione quotidiana permette di impostare la didattica secondo le necessit\ue0 di tutti e di ciascuno, personalizzando gli interventi per gli alunni con Bisogni Educativi Speciali ma rendendoli utili a tutto il contesto sezione. Le strumentazioni digitali a scuola, offrono l\u2019opportunit\ue0 di potenziare le risorse disponibili arricchendo la didattica non solo dal punto di vista dell'accessibilit\ue0 alle discipline, bens\uec dal punto di vista della costruzione di contesti inclusivi. La tecnologia impiegata nello studio di alcune discipline \ue8 sia in grado di integrare l'esperienza dell'apprendimento scolastico, sia capace di supportare anche nuove strategie di cooperative learning. Anche nella scuola dell\u2019infanzia, il ruolo delle tecnologie \ue8 oggetto di indagini per valutare le possibili ricadute sia nei processi di insegnamento-apprendimento, sia nei processi di costruzione di un clima di classe accogliente e inclusivo

Free-hand ultrasound strain elastography in evaluation of soft tissue tumors

Objective: The purpose of this study is to evaluate elastography in a wide spectrum of soft tissue superficial lesions by correlating the elastographic characteristics of these lesions with the elastographic score (ES) system established by Asteria. Methods: Forty patients with different superficial lesions of the soft tissues were studied, including lipomas, schwannomas, neuromas, epidermal inclusion cysts, "in transit" melanoma metastasis, arterio-venous malformation, and giant-cell tumor. An ultrasound examination was performed combined with color-Doppler and elastographic module. The B-mode criteria were echogenicity, margins, and structural homogeneity of the lesion. The color-Doppler criterion was irregular and mainly intra-nodular vascularization. ES 1-4 was attributed, in relation with the increasing tissue stiffness, according to the classification of Asteria adapted for soft tissues. Subsequently, we added to each single B-mode and color-Doppler criterion the ES 3 and 4, thus crossing two parameters of malignancy. All the presumptive diagnoses formulated were confirmed with the clinical data or with the histopathological result. Results: The hypoechoic appearance had the best diagnostic performance. Sensitivity was 87%, specificity 71%, positive predictive value (PPV) 80%, negative predictive value (NPV) 80%, and diagnostic accuracy 80%. There was a good correlation with the clinical and biopsy data, the irregularity of margins the worst performance, the inhomogeneity an intermediate. Color-Doppler had sensitivity 74%, specificity 82%, PPV 85%, NPV 70% and diagnostic accuracy 77.5%. Elastography had sensitivity 87%, specificity 94%, PPV 95%, NPV 84%, and diagnostic accuracy 90%. The combination hypoechoic appearance + ES3/ES4 showed sensitivity 83%, specificity 100%, PPV 100%, NPV 81%,and diagnostic accuracy of 90%. The combination of irregularity of margins + ES3/ES4 showed sensitivity 43%, specificity 100%, PPV 100%, NPV 59%, and diagnostic accuracy of 67.5%. The combination of inhomogeneity of the lesion + ES3/ES4 showed sensitivity 65%, specificity 94%, PPV 94%, NPV 68%, and diagnostic accuracy of 78%. The combination of the color-Doppler with the ES3/ES4 showed sensitivity 69.5%, specificity 100%, PPV 100%, NPV 71%, and diagnostic accuracy of 82.5%.In the combined evaluation, there was a significant increase in specificity, allowing healthy subjects to be categorized as correctly negative, with a reduction in false positives which also translates into an increase in PPV. Conclusions: Elastography alone is not sufficient for a correct diagnostic classification and must be considered as an additional parameter in the study of soft-tissue lesions. Although there was a good agreement between B-mode malignancy criteria and ES3/ES4, there is no significant improvement in sensitivity. Ultrasound assessment, especially of superficial lesions, cannot be separated from an integrated approach that foresees the additional and routine use of the elastographic examination

Diagnostic performance and confidence of contrast-enhanced ultrasound in the differential diagnosis of cystic and cysticlike liver lesions

OBJECTIVE. The aims of this study were to assess the diagnostic performance of contrast-enhanced ultrasound (CEUS) in the characterization of atypical cystic and cysticlike focal liver lesions in comparison with conventional US and to determine whether the use of CEUS can reduce the need for further diagnostic workup. SUBJECTS AND METHODS. In a 3-year period 48 patients with 50 atypical cystic and cysticlike lesions found at conventional US underwent CEUS. Diagnostic confirmation was obtained in cytohistopathologic examinations, with other imaging modalities, and in follow-up. Overall, there were 24 cystic lesions and 26 cysticlike solid lesions, specifically 32 benign and 18 malignant lesions. The conventional US and CEUS images and cine loops were reviewed by two blinded readers independently. Sensitivity, specificity, area under the ROC curve (Az), and interobserver agreement were calculated. RESULTS. Diagnostic performance improved after review of CEUS examinations by both readers (conventional US Az = 0.781 vs 0.972; CEUS Az = 0.734 vs 0.957). Interreader agreement increased, although slightly (conventional US weighted = 0.894; CEUS weighted = 0.953). In terms of differential diagnosis, the occurrence of correctly characterized lesions increased after CEUS for both readers (reader 1, 62% to 98%; reader 2, 56% to 96%). CONCLUSION. The development of low-acoustic-power CEUS has made it possible to identify several imaging features of cystic and cysticlike focal liver lesions that, in association with history and clinical findings, may help to correctly characterize them. Our data indicate the usefulness of CEUS in the evaluation of patients with these lesions

Two interesting cases of Transient Neonatal Diabetes Mellitus

Two cases of transient neonatal diabetes mellitus associated with anemia, macroglossia and umbilical hernia were studied in relation to the possible etiologies that have been postulated to be responsible for this syndrome. Both patients required insulin therapy for the control of their hyperglycemia but case number two needed to be treated for 14 months before glucose normalization occurred. This patient developed classical insulin dependent diabetes mellitus during our follow-up; the HLA typing showed DR4 allele