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Mammalian genomes encode a plethora of long non-coding RNA (lncRNA). These transcripts are thought to regulate gene expression, influencing biological processes from development to pathology. Results from the few lncRNA that have been studied in the context of the immune system have highlighted potentially critical functions as network regulators. Here we explored the nature of the lncRNA transcriptome in regulatory T cells (Tregs), a subset of CD4+ T cells required to establish and maintain immunological self-tolerance. The identified Treg lncRNA transcriptome showed distinct differences from that of non-regulatory CD4+ T cells, with evidence of direct shaping of the lncRNA transcriptome by Foxp3, the master transcription factor driving the distinct mRNA profile of Tregs. Treg lncRNA changes were disproportionally reversed in the absence of Foxp3, with an enrichment for colocalisation with Foxp3 DNA binding sites, indicating a direct coordination of transcription by Foxp3 independent of the mRNA coordination function. We further identified a novel lncRNA Flatr, as a member of the core Treg lncRNA transcriptome. Flatr expression anticipates Foxp3 expression during in vitro Treg conversion, and Flatr-deficient mice show a mild delay in in vitro and peripheral Treg induction. These results implicate Flatr as part of the upstream cascade leading to Treg conversion, and may provide clues as to the nature of this process.

Chronic rhinosinusitis with nasal polyps (CRSwNP) in the severe forms is associated with a poor quality of life. Dupilumab has been suggested as an add-on treatment option for severe CRSwNP. Severe CRSwNP patients treated with Dupilumab in different rhinological units were considered for this study via their evaluation at the baseline at first and the consequential follow-up at 6-, 12-, and 24 months from the first administration. At baseline (T0) and at each follow-up, patients underwent NPS, Sinonasal Outcome Test (SNOT)-22, Visual Analogue Scale (VAS) for smell, and Sniffin' sticks identification test (SSIT). The SNOT-22 domains for function and emotion were also analysed separately. Two hundred and seventeen patients with at least 6 months of follow-up were included. All parameters have improved during treatment ( p < 0.0001). Noticeably, both the function and emotion SNOT-22 domains have improved within 6 months of treatment and have continued to progress during every interval within 12 months from the baseline, positively influencing patients' emotivity and augmenting their social and economic performances. Dupilumab improves the QoL of CRSwNP patients with good effects on the reported productivity and emotional health. Clinicians should pay attention to these two aspects when dealing with patients affected by severe CRSwNP.

Different inflammatory endotypes reflect the heterogeneity of chronic rhinosinusitis with nasal polyps' (CRSwNPs) clinical presentation. This retrospective study aimed to analyze the distribution of polyps in nasal cavities and paranasal sinuses to establish a possible association between CRSwNP endotypes, prognosis, and polyps' extension. This study included 449 adult patients who underwent endoscopic sinus surgery for CRSwNPs between 2009 and 2022. Patients were categorized based on the number of paranasal sinuses involved by polyps. Statistical analyses, including Cox regression, were performed to identify associations between demographic, clinical, and histopathological factors and disease recurrence. CRSwNP patients were stratified into four groups based on the extent of polyp involvement. Asthma and acetylsalicylic acid (ASA) sensitivity were associated with more sinuses involved ( p -values = 0.0003 and 0.0037, respectively). Blood eosinophil counts increased with the number of sinuses affected ( p -value < 0.0001). The distribution of eosinophilic and non-eosinophilic histotypes varied significantly among these groups ( p -value < 0.0001). The risk of CRSwNP recurrence was higher in patients with asthma, higher basophil percentages, and eosinophilic histotype ( p -value 0.0104, 0.0001, 0.0118, and 0.0104, respectively). This study suggests a positive association between the number of paranasal sinuses involved by polyps and the severity of CRSwNPs, particularly in patients with eosinophilic histotype, asthma, and ASA sensitivity.

Aspirin-exacerbated respiratory disease (AERD) is characterized by eosinophilic asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and intolerance to cyclooxygenase-1 inhibitors. Interest is emerging in studying the role of circulating inflammatory cells in CRSwNP pathogenesis and its course, as well as their potential use for a patient-tailored approach. By releasing IL-4, basophils play a crucial role in activating the Th2-mediated response. The main aim of this study was to, first, investigate the level of the pre-operative blood basophils' values, blood basophil/lymphocyte ratio (bBLR) and blood eosinophil-to-basophil ratio (bEBR) as predictors of recurrent polyps after endoscopic sinus surgery (ESS) in AERD patients. The secondary aim was to compare the blood basophil-related variables of the AERD series (study group) with those of a control group of 95 consecutive cases of histologically non-eosinophilic CRSwNP. The AERD group showed a higher recurrence rate than the control group ( p < 0.0001). The pre-operative blood basophil count and pre-operative bEBR were higher in AERD patients than in the control group ( p = 0.0364 and p = 0.0006, respectively). The results of this study support the hypothesis that polyps removal may contribute to reducing the inflammation and activation of basophils.

The prognostic value of conventional histopathological parameters in the sinonasal intestinal-type adenocarcinoma (ITAC) has been debated and novel variables should be investigated. Increasing evidence demonstrated that the evolution of cancer is strongly dependent upon the complex interactions within tumor microenvironment. The aim of this retrospective study was to assess the features of immune microenvironment in terms of CD3 + and CD8 + cells in a series of ITAC and explore their prognostic role, as well as their relations with clinicopathological variables. A computer-assisted image analysis of CD3 + and CD8 + tumor-infiltrating lymphocytes (TIL) density was conducted on surgical specimens of 51 patients with ITAC that underwent a curative treatment including surgery. ITAC displays variable TIL density, which is associated with OS. In a univariate model, the density of CD3 + TIL was significantly related to OS ( p = 0.012), whereas the association with CD8+ TIL density resulted in being non-significant ( p = 0.056). Patients with intermediate CD3 + TIL density were associated with the best outcome, whereas 5-year OS was the lowest for intermediate CD8 + TIL density. CD3 + TIL density maintained a significant association with OS in the multivariable analysis. TIL density was not significantly related to demographic and clinicopathological variables. CD3 + TIL density was independently associated with OS in a non-linear fashion and patients with intermediate CD3 + TIL density had the best outcome. Though based on a preliminary analysis on a relatively small series of patients, this finding makes TIL density a potential independent prognostic factor of ITAC.

Osteoid osteoma (OO) is a primary benign tumor that accounts for up to 3% of all bone tumors. The cervical spine is less affected by OOs, and very few cases of C2 OOs have been reported in the literature, both in adults and children. Surgery may be required in case of functional torticollis, stiffness, and reduced range of motion (ROM) due to cervical OOs refractory to medical therapy. Several posterior and anterior surgical techniques have been described to remove C2 OOs. In particular, anterior approaches to the cervical spine represent the most used surgical route for treating C2 OOs. We describe the first case of OO of the odontoid process removed through a transnasal endoscopic approach with the aid of neuronavigation in a 6-year-old child. No intraoperative complications occurred, and the post-operative course was uneventful. The patient had immediate relief of neck pain and remained pain-free throughout the follow-up period, with complete functional recovery of the neck range of motion (ROM). In this case, based on the favorable anatomy, the transnasal endoscopic approach represented a valuable strategy for the complete removal of an anterior C2 OO without the need for further vertebral fixation since the preservation of ligaments and paravertebral soft tissue.

Background: Retrospectively considering a temporal bone squamous cell carcinomas (TBSCCs) series, our aim was to compare the predictive power of the American Joint Committee on Cancer (AJCC) staging system (8th edition) with the revised Pittsburgh staging system (rPSS) in terms of disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS)., Methods: Forty-three TBSCCs consecutively treated surgically were reviewed. The prognostic performance of AJCC and rPSS was compared., Results: The areas under the curves for the prediction of DFS, DSS, and OS did not differ significantly between both staging systems (p = 0.518, p = 0.940, and p = 0.910, respectively). Harrel's C-indexes for respectively the AJCC and rPSS were 0.76 and 0.70 for DFS, 0.73 and 0.76 for DSS, 0.66 and 0.63 for OS., Conclusion: Comparable prognostic accuracy was observed between AJCC and rPSS. Levels of prognostic performance were only acceptable for both systems, according to Hosmer-Lemeshow scale. Further efforts are needed to define new TBSCC staging modalities with higher prognostic reliability., (© 2022 Wiley Periodicals LLC.)

In the specific field of laryngeal carcinoma (LSCC), evidence about the interaction between angiogenetic pathway and immune microenvironment has not yet been explored. Given the potential relevance of such an interaction for prognostic and therapeutic purposes, the main aim of this exploratory study was to investigate the existence of a correlation between angiogenesis (quantified through CD31 expression), programmed cell death ligand 1 (PD-L1) expression, and immune microenvironment. A secondary aim was to verify whether considering a combination of angiogenesis and immune microenvironment variables might improve prognostic accuracy compared to the traditional clinical-pathological prognostic tools. CD31-assessed micro-vessel density (MVD), PD-L1 in terms of combined positive score (CPS), and tumour infiltrating lymphocytes (TILs) were assessed on 45 consecutive cases of LSCC. Cox proportional hazards model revealed increasing CD31-assessed MVD values, PD-L1 CPS <1, and TILs count rate <30%, as predictive of reduced disease free survival (DFS). Multivariate analysis found that MVD (p<0.0001) and TILs (p=0.0420) retained their significant independent prognostic value. Spearman's correlation model disclosed a significant negative correlation between CD31-assessed MVD values and PD-L1 CPS (p=0.0040). PD-L1 CPS and TILs count rate were positively correlated (p<0.0001). DFS was significantly lower in the CD31-assessed MVD >7, PD-L1 CPS <1, TILs <30% group than in the MVD ≤7, PD-L1 CPS ≥1, TILs ≥30% group (p=0.0001). These data preliminarily support an integrated interpretation of the prognostic role or angiogenesis and immune microenvironment markers in LSCC. This is of potential clinical relevance suggesting a synergistic effect of the combination of anti-angiogenic drugs with programmed death-1/PD-L1 checkpoint inhibitors in advanced LSCC., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Purpose: Our group recently proposed the novel Padova prognostic scoring system for temporal bone carcinoma (TBSCC) that considers: the revised Pittsburgh staging system; radiological dura mater involvement; non-anterior spread (medial, inferior or posterior into the temporal bone and skull base) of T4 tumors; and histological grade. The aim of the present study was to validate this prognostic TBSCC scoring system in a case series selected from the literature., Methods: A search was run to identify studies on TBSCC reporting the variables included in our score for each patient. Then our system was applied to the data extracted., Results: Only two published investigations reported all the clinical and pathological data required for our scoring system. In one series from the Gruppo Otologico in Piacenza (Italy), a significantly higher recurrence rate (p = 0.008), shorter disease-free survival (DFS) (p = 0.001), higher disease-specific mortality (DSM) (p = 0.006), and shorter disease-specific survival (DSS) (p = 0.004) were associated with scores ≥ 5. Receiver operating curve (ROC) analysis showed an AUC of 0.804 for TBSCC recurrence, and 0.832 for DSM. In a series from Kyushu University Hospital (Japan), a significantly higher DSM (p = 0.018) and shorter DSS (p = 0.021) were associated with scores ≥ 5. ROC analysis showed an AUC of 0.812 for tumor relapse and 0.790 for DSM., Conclusion: Our TBSCC Padova scoring system confirmed its validity when applied to the only two international TBSCC series providing the required data. These preliminary results need to be confirmed in a multi-center prospective setting., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: Interactions between SARS-CoV-2 and pharyngeal associated lymphoid tissue are thought to influence the manifestations of COVID-19. We aimed to determine whether a previous history of tonsillectomy, as a surrogate indicator of a dysfunctional pharyngeal associated lymphoid tissue, could predict the presentation and course of COVID-19., Methods: Multicentric cross-sectional observational study involving seven hospitals in Northern and Central Italy. Data on the clinical course and signs and symptoms of the infection were collected from 779 adults who tested positive for SARS-CoV-2, and analysed in relation to previous tonsillectomy, together with demographic and anamnestic data., Results: Patients with previous tonsillectomy showed a greater risk of fever, temperature higher than 39°C, chills and malaise. No significant differences in hospital admissions were found., Conclusions: A previous history of tonsillectomy, as a surrogate indicator of immunological dysfunction of the pharyngeal associated lymphoid tissue, could predict a more intense systemic manifestation of COVID-19. These results could provide a simple clinical marker to discriminate suspected carriers and to delineate more precise prognostic models., (Copyright © 2021 Società Italiana di Otorinolaringoiatria e Chirurgia Cervico-Facciale, Rome, Italy.)

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

This study prospectively assessed the 6-month prevalence of self-reported and psychophysically measured olfactory dysfunction in subjects with mild-to-moderate COVID-19. Self-reported smell or taste impairment was prospectively evaluated by SNOT-22 at diagnosis, 4-week, 8-week, and 6-month. At 6 months from the diagnosis, psychophysical evaluation of olfactory function was also performed using the 34-item culturally adapted University of Pennsylvania Smell Identification Test (CA-UPSIT). 145 completed both the 6-month subjective and psychophysical olfactory evaluation. According to CA-UPSIT, 87 subjects (60.0%) exhibited some smell dysfunction, with 10 patients being anosmic (6.9%) and seven being severely microsmic (4.8%). At the time CA-UPSIT was administered, a weak correlation was observed between the self-reported alteration of the sense of smell or taste and olfactory test scores (Spearman's r = -0.26). Among 112 patients who self-reported normal sense of smell at last follow-up, CA-UPSIT revealed normal smell in 46 (41.1%), mild microsmia in 46 (41.1%), moderate microsmia in 11 (9.8%), severe microsmia in 3 (2.3%), and anosmia in 6 (5.4%) patients; however, of those patients self-reporting normal smell but who were found to have hypofunction on testing, 62 out of 66 had a self-reported reduction in sense of smell or taste at an earlier time point. Despite most patients report a subjectively normal sense of smell, we observed a high percentage of persistent smell dysfunction at 6 months from the diagnosis of syndrome coronavirus 2 (SARS-CoV-2) infection, with 11.7% of patients being anosmic or severely microsmic. These data highlight a significant long-term rate of smell alteration in patients with previous SARS-COV-2 infection., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

GADD45β/MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45β/MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect; however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45β/MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.

Gluten fragments released in gut of celiac individuals activate the innate or adaptive immune systems. The molecular mechanisms associated with the adaptive response involve a series of immunodominant gluten peptides which are mainly recognized by human leucocyte antigen (HLA)-DQ2.5 and HLA-DQ8. Other peptides, such as A-gliadin P31-43, are not recognized by HLA and trigger innate responses by several routes not yet well detailed. Among the gluten fragments known to be active in Celiac disease, here we focus on the properties of all gluten peptides with known tri-dimensional structure either those locked into HLA-DQ complexes whose crystals were X-ray analyzed or characterized in solution as free forms. The aim of this work was to find the structural reasons why some gluten peptides prompt the adaptive immune systems while others do not, by apparently involving just the innate immune routes. We propose that P31-43 is a non-adaptive prompter because it is not a good ligand for HLA-DQ. Even sharing a similar ability to adopt polyproline II structure with the adaptive ones, the way in which the proline residues are located along the sequence disfavors a productive P31-43-HLA-DQ binding.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates., Competing Interests: Declaration of competing interest The authors declare no conflicts of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: This study aimed to develop a novel temporal bone squamous cell carcinoma (TBSCC) prognosis scoring system and compare it with the revised Pittsburgh staging system., Methods: Forty-four consecutive TBSCC patients were assessed to identify predictors of recurrence. Each predictor's hazard ratio for recurrence was used to develop our novel scoring system., Results: Based on variables with P < .10 in Cox's regression model, our score included: revised Pittsburgh stage; non-anterior spread of T4 carcinoma; dural involvement; and histological grade. A higher recurrence rate (P = .000) and shorter disease-free survival (P = .000) were associated with scores of ≥5. The area under the curve of our score was larger than that of the revised Pittsburgh stage for both recurrence and disease-specific mortality (P = .0178 and P = .0193, respectively)., Conclusion: Our TBSCC scoring system is based on variables that are obtainable preoperatively from clinical and radiological data and biopsies. Its prognostic value should be validated for published TBSCC series and then in prospective settings., (© 2020 Wiley Periodicals LLC.)

The ability of many proteins to fold into well-defined structures has been traditionally considered a prerequisite for fulfilling their functions. Protein folding is also regarded as a valuable loophole to escape uncontrolled and harmful aggregations. Here we show that the PBX-regulating protein-1 (PREP1), an important homeodomain transcription factor involved in cell growth and differentiation during embryogenesis, is endowed with an uncommon thermostability. Indeed, circular dichroism analyses indicate that it retains most of its secondary structure at very high temperatures. These findings have important implications for PREP1 functions since it is a stabilizing factor of its partner PBX1. Predictive analyses suggest that the observed PREP1 thermostability could be related to the presence of aggregation-prone regions. Interestingly, synthetic peptides corresponding to these regions exhibit a remarkable propensity to form toxic β-rich amyloid-like aggregates in physiological conditions. On this basis, we suggest that PREP1 stability is an effective way to prevent or limit the formation of harmful aggregates. Notably, one of these PREP1 fragments (residues 117-132) is able to reversibly switch from α-helical to β-rich states depending on the environmental conditions. The chameleon conformational behavior of this peptide makes it an ideal system to study this intriguing and widespread structural transition., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: The interest in surgical routes to the internal auditory canal (IAC) through the external auditory canal for vestibular schwannoma removal has been recently raised by the endoscopic approaches to the lateral skull base. The aim of the study was to reappraise the transmeatal microsurgical approach (TMMa) to the labyrinth and IAC, first described 50 years ago., Methods: A retrospective series of 8 consecutive patients treated for intralabyrinthine and intrameatal schwannomas through TMMa is presented. Main outcome measures consisted of surgical indications, postoperative complications, facial nerve status, bed mobilisation time, hospitalisation time and tumour recurrence rate., Results: Surgical indications for TMMa were tumour growth (62.5%) and disabling vertigo (37.5%) in the present series. Complete tumour removal with no complications and postoperative normal facial nerve function was obtained in all cases. Bed mobilisation occurred after a median of 3 postoperative days (IQR 2.2-3.0) and discharge after a median of 5.6 days (IQR 4.7-7.0). After a median follow-up of 13 months (IQR 7.5-27.5), no tumour recurrence was observed., Conclusions: TMMa indications are limited to schwannomas of the labyrinth and IAC, which dropped out from observation protocols due to unmanageable symptoms or growth. Despite the narrow mini-invasive surgical corridor, the TMMa was a safe an effective microsurgical technique in terms of tumour removal and postoperative course., (Copyright © 2020 Società Italiana di Otorinolaringoiatria e Chirurgia Cervico-Facciale, Rome, Italy.)

There is mounting evidence that a new onset of altered sense of smell or taste is related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In order to allow patients to recognize symptoms indicative of SARS-CoV-2 infection and self-isolate at the earliest opportunity, self-reported loss of smell and taste have greater value in controlling disease transmis- sion than psychophysical testing, which is not widely available outside of highly specialized clinics.

Ribonuclease AS (RNase AS) is a crucial enzyme for virulence of Mycobacterium tuberculosis . We previously observed that RNase AS structurally resembles RNase T from Escherichia coli , an important enzyme for tRNA maturation and turnover. Here, we combine X-ray crystallography and molecular dynamics (MD) to investigate the specificity and dynamic properties of substrate binding. Both X-ray and MD data provide structural determinants that corroborate the strict substrate specificity of RNase AS to cleave only adenosine residues, due to the structural features of adenine base. Beside suggesting tRNA as most likely substrate of RNase AS, MD and modeling studies identify key enzyme-ligand interactions, both involving the catalytic site and the double helix region of tRNA, which is locked by interactions with a set of arginine residues. The MD data also evidence a ligand-induced conformational change of the enzyme which is transferred from one chain to the adjacent one. These data will explain the dimeric nature of both RNase AS and RNase T, with two catalytic grooves composed of both chains. Also, they account for the dichotomy of tRNA, which contains both the substrate poly(A) chain and an inhibiting double strand RNA. Indeed, they provide a possible mechanism of allosteric regulation, which unlocks one catalytic groove when the second groove is inhibited by the double strand region of tRNA. Finally, a full comprehension of the molecular details of tRNA maturation processes is essential to develop novel strategies to modulate RNA processing, for therapeutic purposes. AbbreviationsMDmolecular dynamicsPDBProtein Data BankRMSDroot mean square deviationRMSFroot mean square fluctuationRNAribonucleotidic acidRNase ASRibonuclease ASCommunicated by Ramasamy H. Sarma.

Bicyclic peptides assembled around small organic scaffolds are gaining an increasing interest as new potent, stable and highly selective therapeutics because of their uncommon ability to specifically recognize protein targets, of their small size that favor tissue penetration and of the versatility and easiness of the synthesis. We have here rationally designed bicyclic peptides assembled around a common tri-bromo-methylbenzene moiety in order to mimic the structure of the CFC domain of the oncogene Cripto-1 and, more specifically, to orient in the most fruitful way the hot spot residues H120 and W123. Through the CFC domain, Cripto-1 binds the ALK4 receptor and other protein partners supporting uncontrolled cell growth and proliferation. Soluble variants of CFC have the potential to inhibit these interactions suppressing the protein activity. A CFC analog named B3 binds ALK4 in vitro with an affinity in the nanomolar range. Structural analyses in solution via NMR and CD show that B3 has rather flexible conformations, like the parent CFC domain. The functional effects of B3 on the Cripto-1-positive NTERA cancer cell line have been evaluated showing that both CFC and B3 are cytotoxic for the cells and block the Cripto-1 intracellular signaling. Altogether, the data suggest that the administration of the soluble CFC and of the structurally related analog has the potential to inhibit tumor growth., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Hydrogels produced by self-assembling peptides are intrinsically biocompatible and thus appropriate for many biomedical purposes. Their application field may be even made wider by reducing the softness and improving the hydrogel mechanical properties through cross-linking treatments. To this aim, modifications of EAK16-II sequence by including Cys residues in its sequence were here investigated in order to obtain hydrogels cross-linkable through a disulfide bridge. Two sequences, namely, C-EAK and C-EAK-C, that contain Cys residues at the N-terminus or at both ends were characterized. Fiber-forming abilities and biological and dynamic mechanical properties were explored before and after the oxidative treatment. In particular, the oxidized version of C-EAK presents a good cell viability and sustains osteoblast proliferation. Furthermore, molecular dynamics (MD) simulations on monomeric and assembled forms of the peptides were performed. MD simulations explained how a specific Cys functionalization was better than the other one. In particular, the results suggested that EAK16-II functionalization with a single Cys residue, instead of two, together with biocompatible cross-linking may be considered an intriguing strategy to obtain a support with better dynamic mechanical properties and biological performances.

Inflammation of intestinal tissue in patients affected by celiac disease (CD) originates from the adaptive and innate immune responses elicited by the undigested gliadin fragments through molecular mechanisms not yet completely described. Undigested A-gliadin peptide P31-43 is central to CD pathogenesis, entering enterocytes in vesicular compartments by endocytosis and inducing an innate immune response in CD intestinal mucosa. This study focused on the reasons why P31-43 does not behave as adaptive immunogenic agent. Once obtained by NMR analysis, the three-dimensional model of P31-43 was used to implement a series of in silico experiments aimed to explore the ability of the peptide to interact with HLA-DQ2 and the corresponding receptor onto T cells. Our results show that P31-43 is a poor ligand for DQ2 and/or T-cell receptor. This study was also aimed to investigate, from a structural point of view, the previous experimental findings by which P31-43 is able to enhance the phosphorylation level of the protein ERK2, while some P31-43 Ala-mutants decrease or totally inhibit that process. The molecular models of P31-43, P31-43 P36A, and F37A mutants were used for in silico docking experiments onto the ERK2 structure. The experiments support the hypothesis that P31-43 F37A works as an ERK2 phosphorylation inhibitor because it binds to the ERK2 phosphorylation site. This study reports on the structural properties of so far never NMR characterized gliadin peptides relevant in CD and explores details about their mechanisms of action., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

PASTA domains are small modules expressed in bacteria and found in one or multiple copies at the C-terminal end of several penicillin binding proteins (PBPs) and Ser/Thr protein kinases (STPKs) and represent potential targets for a new class of antibiotics. PASTA domains are currently annotated as sensor domains, as they are thought to activate their cognate proteins in response to binding to opportune ligands. However, recent studies have shown that PASTA domains linked to proteins of different classes, STPKs or PBPs, do not share the same binding abilities. Despite this, there is currently no way to distinguish between PASTA domains from the two classes, since all of them share the same fold, independent of the class they belong to. To identify a predictive tool of class identification, we here analyse a pool of parameters, including amino acid compositions and total charges of PASTA domains either linked to PBPs or to STPKs. We screened sequences from Actinobacteria, Firmicutes and Bacteroidetes. The first two phyla include some of the most dangerous micro-organisms for human health such as Mycobacterium tuberculosis and Staphylococcus aureus. Based on this analysis, our study proposes a predictive method to assign PASTA domains with unknown origin to their corresponding enzyme class, based solely on sequence information.

Aquaporins (AQPs) are among the best structural-characterized membrane proteins, fulfilling the role of allowing water flux across cellular membranes. Thus far, 34 single amino acid polymorphisms have been reported in HUMSAVAR for human aquaporins as disease-related. They affect AQP2, AQP5 and AQP8, where they are associated with nephrogenic diabetes insipidus, keratoderma and colorectal cancer, respectively. For half of these mutations, although they are mostly experimentally characterized in their dysfunctional phenotypes, a structural characterization at a molecular level is still missing. In this work, we focus on such mutations and discuss what the structural defects are that they appear to cause. To achieve this aim, we built a 3D molecular model for each mutant and explored the effect of the mutation on all of their structural features. Based on these analyses, we could collect the structural defects of all the pathogenic mutations (here or previously analysed) under few main categories, that we found to nicely correlate with the experimental phenotypes reported for several of the analysed mutants. Some of the structural analyses we present here provide a rationale for previously experimentally observed phenotypes. Furthermore, our comprehensive overview can be used as a reference frame for the interpretation, on a structural basis, of defective phenotypes of other aquaporin pathogenic mutants.

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them 'mini-factors'. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.

Nodal is a growth factor expressed during early embryonic development, but reactivated in several advanced-stage cancers. Targeting of Nodal signaling, which occurs via the binding to Cripto-1 co-receptor, results in inhibition of cell aggressiveness and reduced tumor growth. The Nodal binding region to Cripto-1 was identified and targeted with a high affinity monoclonal antibody (3D1). By STD-NMR technique, we investigated the interaction of Nodal fragments with 3D1 with the aim to elucidate at atomic level the interaction surface. Data indicate with high accuracy the antibody-antigen contact atoms and confirm the information previously obtained by immune-enzymatic methods. Main residues contacted by 3D1 are P46, V47, E49 and E50, which belong to the Nodal loop involved in the interaction with the co-receptor., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Penicillin binding proteins (PBPs) and Serine Threonine kinases (STPKs) are two classes of bacterial enzymes whose involvement in a series of vital processes in bacterial growth and division is well assessed. Many PBPs and STPKs show linked an ancillary domain named PASTA, whose functional role is not completely deciphered so far. It has been proposed that PASTAs are sensor modules that by binding opportune ligands (i.e. muropeptides) activate the cognate proteins to their functions. However, based on recent data, the sensor annotation sounds true for PASTA from STPKs, and false for PASTA from PBPs., Objective: Different PASTA domains, belonging or not to different protein classes, sharing or not appreciable sequence identities, always show identical folds. This survey of the structural, binding and dynamic properties of PASTA domains pursues the reasons why identical topologies may turn in different roles., Results: Amino acid compositions, total charges and distribution of the hydrophobic/hydrophilic patches on the surface, significantly vary among PASTAs from STPKs and PBPs and appear to correlate with different functions. A possible criterion to discriminate between PASTA modules of STPKs or PBPs solely based on their sequences is proposed. Possibly reflecting different species as well as functional roles and evolutionary profile, our routine represents a fast even though approximate method to distinguish between PASTA belonging to different classes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Glioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. In this context, Bone Morphogenetic Proteins (BMPs) have been demonstrated to induce CSC differentiation and to sensitize GBM cells to treatments., Methods: The BMP-2 mimicking peptide, named GBMP1a, was synthesized on solid-phase by Fmoc chemistry. Structural characterization and prediction of receptor binding were obtained by Circular Dicroism (CD) and NRM analyses. Activation of BMP signalling was evaluated by a luciferase reporter assay and western blot. Pro-differentiating effects of GBMP1a were verified by immunostaining and neurosphere assay in primary glioblastoma cultures., Results: CD and NMR showed that GBMP1a correctly folds into expected tridimensional structures and predicted its binding to BMPR-IA to the same epitope as in the native complex. Reporter analysis disclosed that GBMP1a is able to activate BMP signalling in GBM cells. Moreover, BMP-signalling activation was specifically dependent on smad1/5/8 phosphorylation. Finally, we confirmed that GBMP1a treatment is sufficient to enhance osteogenic differentiation of Mesenchymal Stem Cells and to induce astroglial differentiation of glioma stem cells (GSCs) in vitro., Conclusions: GBMP1a was demonstrated to be a good inducer of GSC differentiation, thus being considered a potential anti-cancer tool to be further developed for GBM treatment., General Significance: These data highlight the role of BMP-mimicking peptides as potential anti-cancer agents against GBM and stimulate the further development of GBMP1a-based structures in order to enhance its stability and activity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide-receptor binding interface were highlighted by docking experiments. Peptide-receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73-92 and 30-34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

Nodal, a member of the TGF-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44-67 of the Nodal protein, corresponding to the pre-helix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here, we show the structural characterization in solution by CD and NMR of the Nodal peptides and the measurement of binding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and binding measurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

Disintegrins, small molecular weight proteins contained in the venom of vipers and rattlesnakes, are high-affinity and selectivity integrin antagonists. Disintegrins inhibitory epitope mainly consists in a tripeptide sequence localized in a mobile loop protruding from the protein core. RTS and/or KTS tripeptide characterizes the most recently discovered group of disintegrins that selectively block α1β1 integrin receptor. A NMR study dedicated to structure and dynamics properties of jerdostatin, an RTS disintegrin, demonstrated that the substitution of the native RTS with KTS motif impaired flexibility and inhibitory activity of the molecule. Here we add atomic details to the experimental profiles of jerdostatin and its R24K mutant by analyzing the dynamics behavior of the molecules through computational methods. For jerdostatin wild type, molecular dynamics simulations and essential dynamics analyses showed that Y31 residue acts as hinge element in the concerted motions involving the active loop and the C-terminal tail. R24 side chain ability to engage both cation-π and H-bond interactions with Y31 residue was found crucial for that breathing mechanism. Less significant loop-tail concerted motions were observed for the R24K mutant. The description at atomic resolution of jerdostatin dynamics is useful for decoding the influence of specific residues on disintegrin functional properties., (© 2014 Wiley Periodicals, Inc.)