Stretching the chains: the destabilizing impact of Cu2+ and Zn2+ ions on K48-linked diubiquitin.

Ubiquitin signalling and metal homeostasis play key roles in controlling several physiological cellular activities, including protein trafficking and degradation. While some relationships between these two biochemical pathways have started to surface, our knowledge of their interplay remains limited. Here, we employ a variety of techniques, such as circular dichroism, differential scanning calorimetry, pressure perturbation calorimetry, fluorescence emission, SDS-PAGE, and small-angle X-ray scattering (SAXS) to evaluate the impact of Cu2+ and Zn2+ ions on the structure and stability of K48 linked diubiquitin (K48-Ub2), a simple model for polyubiquitin chains. The SAXS analysis results show that the structure of the metal-free protein is similar to that observed when the protein is bound to the E2 conjugating enzyme, lending support to the idea that the structure of unanchored K48-linked ubiquitin chains is sufficient for identification by conjugating enzymes without the need for an induced fit mechanism. Our results indicate that K48-Ub2 can coordinate up to four metal ions with both copper and zinc ions inducing slight changes to the secondary structure of the protein. However, we noted significant distinctions in their impacts on protein stability and overall architecture. Specifically, Cu2+ ions resulted in a destabilization of the protein structure, which facilitated the formation of dimer aggregates. Next, we observed a shift in the conformational dynamics of K48-Ub2 toward less compact and more flexible states upon metal ion binding, with Zn2+ inducing a more significant effect than Cu2+ ions. Our structural modelling study demonstrates that both metal ions induced perturbations in the K48-Ub2 structure, leading to the separation of the two monomers thus inhibiting interactions with E2 enzymes. In conclusion, the findings from this study enhance our comprehension of the mechanisms underlying Ub chains recognition. Moreover, they strengthen the notion that drug discovery initiatives aimed at targeting metal-mediated disruptions in Ub signaling hold great potential for treating a wide range of diseases that stem from abnormal protein accumulation.

Idiopathic Non-Dental Facial Pain Syndromes in Italian Children: A Clinical Case Series.

BACKGROUND: The orofacial pain syndromes (OFPs) are a heterogeneous group of syndromes characterized by painful attacks involving the orofacial structures. They may be summarily subdivided into two great categories: (1) orofacial pain mainly attributed to dental disorders such as dentoalveolar and myofascial orofacial pain or temporomandibular joint (TM) pain; (2) orofacial pain mainly attributed to non-dental pain as neuralgias, facial localization of primary headaches or idiopathic orofacial pain. The second group is uncommon, often described by single case reports, can often show overlapping symptoms with the first group, and represents a clinical challenge, carrying the risk of undervaluation and possibly invasive odontoiatric treatment. We aimed to describe a clinical pediatric series of non-dental orofacial pain and better to underline some topographic and clinical features associated with them. We retrospectively collected the data of children admitted to our headache centers (Bari, Palermo, Torino) from 2017 to 2021. Our inclusion criterion was the presence of non-dental orofacial pain following the topographic criteria of 3° International Classification of Headache Disorders (ICHD-3), and exclusion criteria included the pain syndromes attributed to the dental disorders and pain syndromes due to the secondary etiologies Results. Our sample comprised 43 subjects (23/20 M/F, in the range of ages 5-17). We classified them int: 23 primary headaches involving the facial territory during attacks, 2 facial trigeminal autonomic cephalalgias, 1 facial primary stabbing headache, 1 facial linear headache, 6 trochlear migraines, 1 orbital migraine 3 red ear syndrome and 6 atypical facial pain. All patients described debilitating pain for intensity (moderate/severe), 31 children had episodic attacks, and 12 had continuous pain. Almost all received drugs for acute treatment (less than 50% were satisfied), and some received non-pharmacological treatment associated with drug therapy Conclusion. Although rare OFP can occur in pediatric age, it can be debilitating if unrecognized and untreated, affecting the psychophysical well-being of young patients. We highlight the specific characteristics of the disorder for a more correct and earlier identification during the diagnostic process, already difficult in pediatric age, and to define the approach and possible treatment to prevent negative outcomes in adulthood.

Cardiovascular Risk Factors and Family History of Major Thrombotic Events in Children with Migraine: A 12-Year Retrospective Single-Centre Study.

BACKGROUND: Migraine is one of the most frequent primary headaches in childhood. The role of thrombotic predisposition in its pathogenesis is debated. Our aim was to analyse the cardiovascular risk factors and family history of major thrombotic events in children with migraine. METHODS: A retrospective, single-centre study was performed over 12 years. Our headache centre record database was screened for migraine with aura (MA) and migraine without aura (MO) on the basis of the ICHD-II (until 2013) and III criteria. A control group of otherwise healthy children was recruited. Descriptive and multivariate analyses are provided; significance was set at p < 0.05. RESULTS: Migraine was diagnosed in 930 children (24.7% MA); 73.3% were 9-14 years old. Children with MA were older (p < 0.001). A family history of cerebral ischemic events at ≤50 years old was more commonly reported by children with MA than those with MO (p < 0.001) and those in the control group (p = 0.001). Children with MA showed a higher risk of a family history of cerebral ischemic events at ≤50 years old than children with MO (OR: 2.6) and those in the control group (OR: 3.1). When comparing the family history of DVT, we observed a significantly increased risk for MA vs. MO (OR: 2.9). CONCLUSION: A family history of cerebral ischemic events at ≤50 years old leads to an increased risk of MA. Further studies are needed to explore such an association.

Interleukin 9 neutralisation reduces collagen-induced arthritis severity in mouse models.

OBJECTIVES: Interleukin 9 (IL-9) is a mediator of tissue damage in several inflammatory diseases. In this study we aimed to evaluate the effects of in vivo IL-9 neutralisation in mice developing collagen induced arthritis (CIA). METHODS: DBA/1 were immunised with collagen in Freund's complete adjuvant (CFA) to induce arthritis. Anti-IL-9 mAb was injected in mice after the onset of arthritis (Group A) or on the same day as sensitisation and again on the day of the challenge (Group B). Histological analysis was performed in joints of mice and spleen cells were also analysed by flow cytometry. A geneset analysis was carried out on whole tarsal joint tissue transcriptomes. RESULTS: IL-9 was over-expressed in swollen joints of mice developing arthritis. Treatment with anti-IL-9 mAb after arthritis onset efficiently down-modulated the severity of joint inflammation. Similarly, anti-IL-9 mAb administered on the same day as sensitisation and on the day of challenge also delayed the onset of arthritis. Anti-IL-9 mAb injection after the onset of arthritis was associated with a decrease of CD4+ TNF-α+ cells and an increase of CD4+ FoxP3+ IL-10+ cells. Geneset analysis in CIA showed an up-regulation of GATA3 with no significant direct interactions between IL-9 and GATA3, which instead was mediated by IL-5 through STAT6. CONCLUSIONS: Our results suggest that IL-9 is involved in the immunopathogenesis of CIA. Further implications for the clinical translation of our findings are discussed.

Microbiome Changes in Connective Tissue Diseases and Vasculitis: Focus on Metabolism and Inflammation.

The microbial community acts as an active player in maintaining homeostasis and immune functions through a continuous and changeable cross-talk with the host immune system. Emerging evidence suggests that altered microbial composition, known as dysbiosis, might perturb the delicate balance between the microbiota and the immune system, triggering inflammation and potentially contributing to the pathogenesis and development of chronic inflammatory diseases. This review will summarize the current evidence about the microbiome-immunity cross-talk, especially focusing on the microbiota alterations described in patients with rheumatic diseases and on the recent findings concerning the interaction between microbiota, metabolic function, and the immune system.

Role of the IL-23/IL-17 Pathway in Rheumatic Diseases: An Overview.

Interleukin-23 (IL-23) is a pro-inflammatory cytokine composed of two subunits, IL-23A (p19) and IL-12/23B (p40), the latter shared with Interleukin-12 (IL-12). IL-23 is mainly produced by macrophages and dendritic cells, in response to exogenous or endogenous signals, and drives the differentiation and activation of T helper 17 (Th17) cells with subsequent production of IL-17A, IL-17F, IL-6, IL-22, and tumor necrosis factor α (TNF-α). Although IL-23 plays a pivotal role in the protective immune response to bacterial and fungal infections, its dysregulation has been shown to exacerbate chronic immune-mediated inflammation. Well-established experimental data support the concept that IL-23/IL-17 axis activation contributes to the development of several inflammatory diseases, such as PsA, Psoriasis, Psoriatic Arthritis; AS, Ankylosing Spondylitis; IBD, Inflammatory Bowel Disease; RA, Rheumatoid Arthritis; SS, Sjogren Syndrome; MS, Multiple Sclerosis. As a result, emerging clinical studies have focused on the blockade of this pathogenic axis as a promising therapeutic target in several autoimmune disorders; nevertheless, a greater understanding of its contribution still requires further investigation. This review aims to elucidate the most recent studies and literature data on the pathogenetic role of IL-23 and Th17 cells in inflammatory rheumatic diseases.

The interplay between lipid and Aß amyloid homeostasis in Alzheimer's Disease: risk factors and therapeutic opportunities.

Alzheimer's Diseases (AD) is characterized by the accumulation of amyloid deposits of Aß peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aß deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable. Recently, many studies have described the key role played by membrane bound Aß assemblies in neurotoxicity. Moreover, it has been suggested that a derangement of the ubiquitin proteasome pathway and autophagy is significantly correlated with toxic Aß aggregation and dysregulation of lipid levels. Thus, studies focusing on the role played by lipids in Aß aggregation and proteostasis could represent a promising area of investigation for the design of valuable treatments. In this review we examine current knowledge concerning the effects of lipids in Aß aggregation and degradation processes, focusing on the therapeutic opportunities that a comprehensive understanding of all biophysical, biochemical, and biological processes involved may disclose.

The Role of Copper (II) on Kininogen Binding to Tropomyosin in the Presence of a Histidine-Proline-Rich Peptide.

The antiangiogenic activity of the H/P domain of histidine-proline-rich glycoprotein is mediated by its binding with tropomyosin, a protein exposed on endothelial cell-surface during the angiogenic switch, in presence of zinc ions. Although it is known that copper ion serum concentration is significantly increased in cancer patients, its role in the interaction of H/P domain with tropomyosin, has not yet been studied. In this paper, by using ELISA assay, we determined the modulating effect of TetraHPRG peptide, a sequence of 20 aa belonging to H/P domain, on the binding of Kininogen (HKa) with tropomyosin, both in absence and presence of copper and zinc ions. A potentiometric study was carried out to characterize the binding mode adopted by metal ions with TetraHPRG, showing the formation of complex species involving imidazole amide nitrogen atoms in metal binding. Moreover, circular dichroism showed a conformational modification of ternary systems formed by TetraHPRG, HKa and copper or zinc. Interestingly, slight pH variation influenced the HKa-TetraHPRG-tropomyosin binding. All these results indicate that both metal ions are crucial in the interaction between TetraHPRG, tropomyosin and HKa.

Primary Sjogren Syndrome: Focus on Innate Immune Cells and Inflammation.

Primary Sjogren Syndrome (pSS) is a complex, multifactorial rheumatic disease that mainly targets salivary and lacrimal glands, inducing epithelitis. The cause behind the autoimmunity outbreak in pSS is still elusive; however, it seems related to an aberrant reaction to exogenous triggers such as viruses, combined with individual genetic pre-disposition. For a long time, autoantibodies were considered as the hallmarks of this disease; however, more recently the complex interplay between innate and adaptive immunity as well as the consequent inflammatory process have emerged as the main mechanisms of pSS pathogenesis. The present review will focus on innate cells and on the principal mechanisms of inflammation connected. In the first part, an overview of innate cells involved in pSS pathogenesis is provided, stressing in particular the role of Innate Lymphoid Cells (ILCs). Subsequently we have highlighted the main inflammatory pathways, including intra- and extra-cellular players. A better knowledge of such processes could determine the detection of new therapeutic targets that are a major need for pSS.

The Ionophoric Activity of a Pro-Apoptotic VEGF165 Fragment on HUVEC Cells.

Copper plays an important role as a regulator in many pathologies involving the angiogenesis process. In cancerogenesis, tumor progression, and angiogenic diseases, copper homeostasis is altered. Although many details in the pathways involved are still unknown, some copper-specific ligands have been successfully used as therapeutic agents. Copper-binding peptides able to modulate angiogenesis represent a possible way to value new drugs. We previously reported that a fragment (VEGF73-101) of vascular endothelial growth factor (VEGF165), a potent angiogenic, induced an apoptotic effect on human umbilical vein endothelial cells. The aim of this study was to investigate the putative copper ionophoric activity of VEGF73-101, as well as establish a relationship between the structure of the peptide fragment and the cytotoxic activity in the presence of copper(II) ions. Here, we studied the stoichiometry and the conformation of the VEGF73-101/Cu(II) complexes and some of its mutated peptides by electrospray ionization mass spectrometry and circular dichroism spectroscopy. Furthermore, we evaluated the effect of all peptides in the absence and presence of copper ions by cell viability and cytofuorimetric assays. The obtained results suggest that VEGF73-101 could be considered an interesting candidate in the development of new molecules with ionophoric properties as agents in antiangiogenic therapeutic approaches.

Hyaluronic acid and platelet-rich plasma, a new therapeutic alternative for scleroderma patients: a prospective open-label study.

BACKGROUND: Systemic sclerosis is a systemic connective tissue disease characterized by endothelium damage, fibrosis, and subsequent atrophy of the skin. Perioral fibrosis produces a characteristic microstomia together with microcheilia, both of which cause severe difficulties and affects patients' daily life, such as eating and oral hygiene. Since there are no effective and specific therapies, we have aimed at evaluating the response to filler injections of hyaluronic acid together with platelet-rich plasma. METHODS: Ten female patients aged between 18 and 70 were included in this study. Each patient was treated with three filler injections of hyaluronic acid and platelet-rich plasma at an interval of 15 to 20 days. Follow-up check-ups were recorded 1, 3, and 24 months after the end of the treatment. During the therapy and the subsequent follow-up, we evaluated the mouth's opening, freedom of movement of the lips, and skin elasticity. RESULTS: After the treatment, patients had achieved good results already after the first injection and the improvement was maintained in the following months, up to 2 years. In particular, 8 (80%) patients showed a greater mouth's opening and increased upper lip's thickness during 1-month follow-up and maintained these results after 2 years (maximum mouth's opening T0 47.61; T3 49.23; T4 48.60 p <  0.0001. Upper lip's thickness T0 4.20; T3 4.75; T4 4.45 p <  0.0001). Moreover, distance between upper and lower incisors (T0 27.05; T3 29.03; T4 28.14 p < 0.0001), inter-commissural distance (T0 49.12; T3 51.44; T4 50.31: p < 0.0001), and lower lip's thickness (T0 3.80; T3 4.85, 5.10; T4 4.25; p < 0.0001) were increased in all of patients in 1-month follow-up, keeping these benefits after 24 months and having a significant increase of skin elasticity 1 month after the end of therapy. CONCLUSIONS: Our study demonstrates that filler injections of hyaluronic acid and platelet-rich plasma represent an efficient local therapeutic alternative for patients affected by scleroderma. The treatment has significantly improved patients' quality of living.

Peptides derived from the histidine-proline rich glycoprotein bind copper ions and exhibit anti-angiogenic properties.

The role of copper in cancer progression has been established since decades. Additionally, copper is able to stimulate angiogenesis through the control of VEGF expression and activity in endothelial cells. In this paper a tetrapeptide, belonging to the histidine-proline-rich glycoprotein (HPRG) and encompassing four repeats of the sequence GHHPH (named TetraHPRG), was synthesized and its copper(ii) complex species were characterized by means of potentiometry, UV-vis, circular dichroism (CD), electron paramagnetic resonance (EPR) and electron spray ionization mass spectrometry (ESI-MS). Moreover, a peptide covalently bound through an amidic bond to trehalose (TH-TetraHPRG) was designed and synthesized as a prodrug system. The activity of both TetraHPRG and TH-TetraHPRG molecules on copper and VEGF induced angiogenic responses in endothelial cells was assessed. The two peptides show a similar and effective anti-angiogenic activity on both molecular and cellular responses. Since the trehalose derivative has a higher resistance to enzymatic degradation, it can be further exploited as a potential drug delivery system with anti-angiogenic activity.

Water soluble glucose derivative of thiocarbohydrazone acts as ionophore with cytotoxic effects on tumor cells.

A novel water-soluble ionophore based on the thiocarbohydrazone moiety conjugated with glucose (GluTch) was synthesized through a simple two-step procedure. Structural elucidation was carried out in water solution by means of various spectroscopic techniques (NMR, UV-Vis, and CD), electrospray ionization mass spectrometry and density functional theory calculations. The flexible nature of the thiocarbohydrazone moiety of the new glycoderivative compound induced both different coordination motifs and stoichiometry towards copper and zinc. Cytotoxicity assays of the ligands on the human normal keratinocyte NCTC-2544, MDA-MB-231 breast cancer and PC-3 human prostate adenocarcinoma cell lines demonstrated that i) higher activity on cancer cells growth inhibition compared to a normal cell line; ii) the introduction of the glucose unit does not alter the cytotoxic activity of the underivatized ionophore ligand and iii) the presence of copper ion improves the activity of the thiocarbohydrazones.

Headache in the pediatric emergency department: A 5-year retrospective study.

Aim To determine the red flags for serious organic causes of headache in children, to analyze if the management of headache in the Pediatric Emergency Department is appropriate, and whether the follow-up may limit repeated visits to the Emergency Department. Methods All the patients ≤ 18 years referred to our pediatric Emergency Department for non-traumatic headache over 5 years were retrospectively reviewed. The patients followed up by the Pediatric Headache Centre were also screened. Statistical analysis was undertaken using the Chi-squared test or Fisher's exact test and multivariate analysis; significance at p < 0.05. Results 1833 patients (54.6% males) accessed our Emergency Department 2086 times; 62.1% had primary headache, 30.0% had secondary headache, 7.8% received inconsistent diagnosis. Among those with secondary headache, 24 (1.1% of total visits) were diagnosed with serious disorders. The clinical red flags for "serious headache" were: Cranial nerves palsy, strabismus, and drowsiness. One hundred and eighty four patients (8.8 %) underwent neuroimaging (rate of pathological findings: 7.1 %); 37.2 % of the patients received analgesic therapy. One hundred and fifteen patients (6.2 %) returned within three months; 24 of these were referred to the Headache Centre, with only one accessing the Emergency Department again. Conclusions The vast majority of headaches referred to the Pediatric Emergency Department are benign, and primary forms prevail. "Serious headache" is rare and shows typical clinical features and abnormal neurologic evaluation; specific clinical red flags, along with suggestive personal history, should lead the pediatrician to prescribe only appropriate neuroimaging. Pain relief is still insufficient in the Pediatric Emergency Department despite appropriate guidelines. Last, the collaboration with the Headache Centre is crucial to limit repeated visits.

The Inorganic Perspective of VEGF: Interactions of Cu(2+) with Peptides Encompassing a Recognition Domain of the VEGF Receptor.

The vascular endothelial growth factor A (VEGF-A) is a potent angiogenic factor, its activity may be influenced by the presence of copper(II) ions. To mimic the interaction between copper(II) and VEGF (Vascular Endotelial Growth Factor), the N- and C-terminally blocked peptide fragments VEGF73-101 and VEGF84-101, owing to VEGF165 protein, have been synthesized. These protein domains represent a specific recognition site with the VEGF receptor (VEGFR). Copper(II) complexes with VEGF73-101 and VEGF84-101 were investigated by means of potentiometry and UV-Vis, ESI-MS, CD, EPR spectroscopic methods. Both peptides have three histidine residues and display a binding high affinity for copper(II) ions. The proliferative activity of the peptides in the absence and presence of copper(II) ions as well as of VEGF-165 protein was also tested on HUVEC cells (Human Umbilical Vein Endothelial Cells). The VEGF73-101 showed a dose-dependent anti-proliferative activity, while the shorter peptide VEGF84-101 did not affect HUVEC proliferation, both in the presence and in the absence of VEGF.

Inorganic stressors of ubiquitin.

Many neurodegenerative proteinopathies are characterized by ubiquitin (Ub)-containing intraneuronal inclusion bodies. Recent reports have shown that Ub is able to bind Cu(II) and Zn(II), the dyshomeostasis of which is a hallmark of neurodegeneration. Here we use complementary techniques like potentiometry, circular dichroism-visible, and electron spin resonance to unveil the Ub/metal species that form, at neutral pH, their binding constants and structural features. Next, we show that both Zn(II) and Cu(II) ions hinder the interactions between Ub and Ub-conjugating E2 enzymes and inhibit significantly both Lys48 and Lys63 self-polyubiquitination reactions in a cell-free medium. The effects of Zn(II) and Cu(II) on Lys63 and Lys48 polyUb chain synthesis are compatible with the hypothesis that metal binding to His68 modifies the Ile44 hydrophobic patch of Ub and makes the protein less available for polyUb. These findings contribute to further arguments for a close relationship between metal dyshomeostasis and abnormal protein degradative pathways in the upstream events, triggering neurodegeneration.

Synthesis, chemical characterization and biological activity of new histone acetylation/deacetylation specific inhibitors: a novel and potential approach to cancer therapy.

Three new triorganotin(IV) complexes of valproic acid (vp1, Me3Sn-valproate; vp2, Bu3Sn-valproate; vp3, Ph3Sn-valproate) have been synthesized and investigated by spectroscopic and biological methods. An anionic, monodentate valproate ligand was observed, ester-like coordinating the tin atom on a tetra-coordinated, monomeric environment. The structures, though, can distort towards a penta-coordination, as a consequence of a long range O···Sn interaction. Crystallographic and NMR findings confirm this situation both in solid state and solution. Biological finding evidenced a clear cytotoxic action of the complexes in hepatocellular carcinoma cell cultures: one of the complexes induced an 80% cell viability reduction after 24h treatment in HepG2 cells. This effect was accompanied by the appearance of biochemical signs of apoptosis. In Chang liver cells, the same compound induced only modest effects, suggesting a potential use as anti-cancer drug. Preliminary evaluations on hyperacetylation state of histone H3 in tributyltin-valproate treated HepG2 cells showed an increase in Ac-H3 (histone H3 acetylated at lys-9 and lys-14), suggesting that the compound maintains the deacetylation inhibition activity of its ligand valproate.

Prognostic values of multichannel intraluminal impedance and pH monitoring in newborns with symptoms of gastroesophageal reflux disease.

OBJECTIVE: To evaluate the relationship between multichannel intraluminal impedance and pH monitoring (MII/pH) values in newborns with symptoms of gastroesophageal reflux disease (GERD) and clinical history in their first 3 years of life. STUDY DESIGN: Sixty-four newborns with GERD symptoms who underwent MII/pH in the first weeks of life were enrolled into a clinical follow-up program. Follow-up visits were programmed at 1, 3, 6, 9, 12, 18, 24, and 36 months. Patients were divided into 3 groups according to duration of symptoms: short (1-3 months), medium (4-9 months), and long (>9 months), and MII/pH values in these groups were compared. RESULTS: Fifty-three patients completed the 3-year follow-up. The number of patients with GERD symptoms decreased each month. A comparison of MII/pH values of the 3 lifetime symptom groups revealed differences in the impedance bolus exposure index (F = 83; P = .012) and proximal reflux frequency (F = 410; P = .022). These 2 MII variables showed an increasing trend from the short lifetime symptom group to the long lifetime symptom group. Weakly acidic reflux events, but not acidic events, were responsible for these differences. CONCLUSION: MII/pH in newborns has prognostic value regarding the duration of GERD symptoms and provides useful information that clinicians may give parents about the prognosis of symptomatic infants. Impedance bolus exposure index and proximal reflux frequency seem to be the variables with the highest predictive value. Weakly acidic reflux events play an important role in determining the duration of GERD symptoms in newborns.