Protocol for photo-controlling the assembly of cyclic peptide nanotubes in solution and inside microfluidic droplets.

In this protocol, we describe how to perform the photo-isomerization of cyclic peptides containing an unsaturated ß-amino acid. This process triggers the formation or disassembly of cyclic peptide nanotubes under appropriate light irradiation. Specifically, we start by describing the solid-phase synthesis of the cyclic peptide component. We also present a technique for performing isomerization studies in solution and how to extend it to microfluidic aqueous droplets. For complete details on the use and execution of this protocol, please refer to Vilela-Picos et al.1.

Prevalence of Long COVID Symptoms Related to SARS-CoV-2 Strains.

BACKGROUND: Few studies have assessed the differences of patterns of Long COVID (L-COVID) with regards to the pathogenetic SARS-CoV-2 strains. OBJECTIVES: To investigate the relationship between demographic and clinical characteristics of acute phase of infection and the persistence of L-COVID symptoms and clinical presentation across different SARS-CoV-2 strains. METHODS: In this observational-multicenter study we recorded all demographic and clinical characteristics, severity of infection, presence/persistence of symptoms of fatigue, dyspnoea and altered quality of life (QoL) at baseline and after 6 months, in a sample of Italian patients from Liguria between March 2020 and March 2022. RESULTS: 308 patients (mean age 63.2 years; 55.5% men) with previous COVID were enrolled. Obese patients were 21.2% with a significant difference in obesity prevalence across the second and third wave (p = 0.012). Treatment strategies differed between waves (p < 0.001): more patients required invasive mechanical ventilation in the first wave, more patients were treated with high-flow nasal cannula/non-invasive ventilation in the in the second and more patients were treated with oxygen-therapy in the fourth wave. At baseline, a high proportion of patients were symptomatic (dyspnoea and fatigue), with impairment in some QoL indicators. A higher prevalence of patients with pain, were seen in the first wave compared to later infections (p = 0.01). At follow-up, we observed improvement of dyspnoea, fatigue and some dimensions of QoL scale evaluation such as mobility, usual activities, pain evaluations; instead there was no improvement in remaining QoL scale indicators (usual care and anxiety-depression). CONCLUSIONS: There were no significant differences in the prevalence of the most frequent L-COVID symptoms, except for QoL pain domain that was especially associated with classical variant. Our results show substantial impact on social and professional life and usual care activities. These findings highlight the importance of multidisciplinary post COVID follow-up care including mental health support and rehabilitation program.

Prognostic significance of peripheral consolidations at chest x-ray in severe COVID-19 pneumonia.

To evaluate the possible prognostic significance of the development of peripheral consolidations at chest x-ray in COVID-19 pneumonia, we retrospectively studied 92 patients with severe respiratory failure (PaO2/FiO2 ratio < 200 mmHg) that underwent at least two chest x-ray examinations (baseline and within 10 days of admission). Patients were divided in two groups based on the evolution of chest x-ray toward the appearance of peripheral consolidations or toward a greater extension of the lung abnormalities but without peripheral consolidations. Patients who developed lung abnormalities without peripheral consolidations as well as patients who developed peripheral consolidations showed, at follow-up, a significant worsening of the PaO2/FiO2 ratio but a significantly lower mortality and intubation rate was observed in patients with peripheral consolidations at chest x-ray. The progression of chest x-ray toward peripheral consolidations is an independent prognostic factor associated with lower intubation rate and mortality.

Molecular Plumbing to Bend Self-Assembling Peptide Nanotubes.

Light-induced molecular piping of cyclic peptide nanotubes to form bent tubular structures is described. The process is based on the [4+4] photocycloaddition of anthracene moieties, whose structural changes derived from the interdigitated flat disposition of precursors to the corresponding cycloadduct moieties, induced the geometrical modifications in nanotubes packing that provokes their curvature. For this purpose, we designed a new class of cyclic peptide nanotubes formed by ß- and α-amino acids. The presence of the former predisposes the peptide to stack in a parallel fashion with the ß-residues aligned along the nanotube and the homogeneous distribution of anthracene pendants.

Double Orthogonal Click Reactions for the Development of Antimicrobial Peptide Nanotubes.

A new class of amphipathic cyclic peptides, which assemble in bacteria membranes to form polymeric supramolecular nanotubes giving them antimicrobial properties, is described. The method is based on the use of two orthogonal clickable transformations to incorporate different hydrophobic or hydrophilic moieties in a simple, regioselective, and divergent manner. The resulting cationic amphipathic cyclic peptides described in this article exhibit strong antimicrobial properties with a broad therapeutic window. Our studies suggest that the active form is the nanotube resulted from the parallel stacking of the cyclic peptide precursors. Several techniques, CD, FTIR, fluorescence, and STEM, among others, confirm the nanotube formation.

Polymorphic Self-Organization of Lauroyl Peptide in Response to pH and Concentration.

Amphipathic peptides are attractive building blocks for the preparation of self-assembling, bio-inspired, and stimuli responsive nanomaterials with pharmaceutical interest. The bioavailability of these materials can be improved with the insertion of d amino acid residues to avoid fast proteolysis in vivo. With this knowledge, a new lauroyl peptide consisting of a sequence of glycine, glycine, d-serine, and d-lysine was designed. In spite of its simple sequence, this lipopeptide self-assembles into spherical micelles at acid pH, when the peptide moiety adopts disordered conformations. Self-aggregates reshape toward fibers at basic pH, following the conformational transition of the peptide region from random coil to ß-sheet. Finally, hydrogels are achieved at basic pH and higher concentrations. The transition from random coil to ß-sheet conformation of the peptide headgroup obtained by increasing pH was monitored by circular dichroism and vibrational spectroscopy. A structural analysis, performed by combining dynamic light scattering, small-angle X-ray scattering, transmission electron microscopy, and molecular dynamic simulations, demonstrated that the transition allows the self-assemblies to remodel from spherical micelles to rodlike shapes, to long fibers with rectangular cross-section and a head-tail-tail-head structure. The viscoelastic behavior of the hydrogels formed at the highest pH was investigated by rheology measurements.

Switchable length nanotubes from a self-assembling pH and thermosensitive linear l,d-peptide-polymer conjugate.

Preparation and characterization of a pH and thermosensitive linear l,d-octapeptide-poly(dimethylamino ethyl methacrylate) ((l-Val-d-Val)4-PDMAEMA) conjugate is reported. The hydrophobic uncharged linear (l-Val-d-Val)4 octapeptide was designed to self-assemble in nanotubes by exploiting the tubular self-assembling properties of linear peptides with regularly alternating enantiomeric sequences. pH and thermosensitive PDMAEMA was obtained by atom transfer radical polymerization (ATRP). The conjugate was prepared by click-chemistry on the solid phase synthetized peptide. Because of the strong interactions between the peptide moieties, long single channel nanotubes (0.2-1.5 µm) are formed also at acidic pH with the fully charged polymer. At 25 °C and basic pH the size of the nanotubes did not change significantly. In basic conditions and temperature above the PDMAEMA lower critical solution temperature (LCST) a significant increase of the length of the nanotubes up to several micrometers is observed. The size is retained for several days after cooling back to room temperature. Sonication significantly reduces the nanotube length (0.2-0.5 µm) forming low polydisperse nanotubes. The elongation of the nanotubes is fully reversible by restoring acidic pH. This is the first example, to our knowledge, of thermosensitive peptide-polymer single channel nanotubes with length that can be varied from hundreds of nanometers to several micrometers.

Comorbidities are associated with different features of severe asthma.

BACKGROUND: According to ATS/ERS document on severe asthma (SA), the management of these patients requires the identification and proper treatment of comorbidities, which can influence the control of asthma. METHODS: The aim of this study was to assess the independent effect of different comorbidities on clinical, functional and biologic features of SA. Seventy-two patients with SA according to GINA guidelines were examined. We collected demographic data, smoking habit, asthma history, and assessment of comorbidities. Pulmonary function, inflammatory biomarkers, upper airway disease evaluation, asthma control and quality of life were carefully assessed. RESULTS: The mean age of patients was 59.1 years (65.3% female, 5.6% current smokers). Comorbidities with higher prevalence were: chronic rhinosinusitis with or without nasal polyps (CRSwNP or CRSsNP), obesity and gastro-esophageal reflux (GERD), with some overlapping among them. In an univariate analysis comparing patients with single comorbidities with the other ones, asthmatics with CRSwNP had lower lung function and higher sputum eosinophilia; obese asthmatics had worse asthma control and quality of life, and tended to have lower sputum eosinophils; asthmatics with GERD showed worse quality of life. In multivariate analysis, obesity was the only independent factor associated with poor asthma control (OR 4.9), while CRSwNP was the only independent factor associated with airway eosinophilia (OR 16.2). Lower lung function was associated with the male gender and longer duration of asthma (OR 3.9 and 5.1, respectively) and showed a trend for the association with nasal polyps (OR 2.9, p = 0.06). CONCLUSION: Our study suggests that coexisting comorbidities are associated with different features of SA.

Can the response to Omalizumab be influenced by treatment duration? A real-life study.

OBJECTIVE: It is unknown whether Omalizumab effectiveness changes over the course of time. Our retrospective real-life study tried to analyze whether Omalizumab response may be influenced by treatment duration. METHODS: 340 severe asthmatics treated with Omalizumab for different periods of time were recruited. They were subdivided into 4 groups according to the Omalizumab treatment length: <12, between 12 and 24, between 24 and 60 and >60 months. Omalizumab treatment results (FEV1, exacerbations, ACT, SABA use, asthma control levels, medications used e and ICS doses) were compared. RESULTS: ACT, exacerbations, GINA control levels, ICS doses and SABA use were similar in all groups with different Omalizumab treatment durations. Using a linear regression model, corrected for all confounding variables, a higher significant positive increase in FEV1% in subjects treated for 12-24 (ß = 9.49; p = 0.034) or 24-60 months (ß = 8.56; p = 0.043) was found when compared with subjects treated for a shorter period. Treatment duration was positively associated with a step down of the other associated therapies (OR: 1.013; p = 0.019). This association was more relevant (OR: 4.167; p = 0.005) when we considered Omalizumab treatment duration >60 months compared to the shorter therapy. In particular, the percentage of subjects that were taking Montelukast, LABAs and oral corticosteroids was lower in the group treated with Omalizumab for a longer period of time. CONCLUSION: In real-life, the positive Omalizumab response remained stable for over 60 months. Long term Omalizumab treatment may lead to a discontinuation of some associated medications and to a slowing down of FEV1 decline.

Benzimidazole-based derivatives as privileged scaffold developed for the treatment of the RSV infection: a computational study exploring the potency and cytotoxicity profiles.

Respiratory syncytial virus (RSV) has been identified as a main cause of hospitalisation in infants and children. To date, the current therapeutic arsenal is limited to ribavirin and palivizumab with variable efficacy. In this work, starting from a number of in-house series of previously described anti-RSV agents based on the benzimidazole scaffold, with the aim at gaining a better understanding of the related chemical features involved in potency and safety profiles, we applied a computational study including two focussed comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The results allowed us to derive useful suggestions for the design of derivatives and also to set up statistical models predicting the potency and selectivity index (SI = CC50/EC50) of any new analogue prior to synthesis. Accordingly, here, we discuss preliminary results obtained through the applied exhaustive QSAR analyses, leading to design and synthesise more effective anti-RSV agents.

New arylsparteine derivatives as positive inotropic drugs.

Positive inotropic agents are fundamental in the treatment of heart failure; however, their arrhythmogenic liability and the increased myocardial oxygen demand strongly limit their therapeutic utility. Pursuing our study on cardiovascular activities of lupin alkaloid derivatives, several 2-(4-substituted-phenyl)-2-dehydrosparteines and 2-(4-substituted-phenyl)sparteines were prepared and tested for inotropic and chronotropic activities on isolated guinea pig atria. Four compounds (6b, 6e, 7b, and 7f) exhibited significant inotropism that, at the higher concentrations, was followed by negative inotropism or toxicity. Compound 7e (2-(4-tolyl)sparteine) exhibited a steep dose-depending inotropic activity up to the highest concentration tested (300 µM) with an Emax of 116.5 ± 3.4% of basal force, proving less potent but much more active in comparison to the highest concentrations tested of digoxin and milrinone having Emax of 87.5 ± 3.1% and 52.2 ± 1.1%, respectively. Finally, docking studies suggested that the relevant sparteine derivatives could target the sigma-1 receptor, whose involvement in cardiac activity is well documented.

PEGylated ß-Sheet Breaker Peptides as Inhibitors of ß-Amyloid Fibrillization.

Three PEGylated ß-sheet breaker peptides are designed as new inhibitors of ß-amyloid fibrillization. The peptide Ac-Leu-Pro-Phe-Phe-Asp-NH2 , considered the lead compound, and hexamers in which taurine and ß-alanine substitute the acetyl group, are conjugated to poly(ethylene glycol); this conjugates self-assemble into nanoparticles. The activity of the PEGylated peptides as inhibitors of amyloid fibrillization are tested in vitro using circular dichroism spectroscopy and scanning electron microscopy. The experimental results indicate that PEGylation does not impair the ability of the ß-sheet breaker peptides to inhibit fibrillogenesis in vitro. Moreover, microscopy images of ß-amyloid incubated for 6 days with the taurine-containing peptide, suggest that this conjugate has major anti-fibrillogenesis activity and demonstrate the important role of the sulfonamide function against the amyloid aggregation.

Self-assembly and drug release study of linear l,d-oligopeptide-poly(ethylene glycol) conjugates.

The preparation and structural organisation of new bioinspired nanomaterials based on regular alternating enantiomeric sequence of tetra- and hexapeptides end-linked to poly(ethylene glycol) (PEG) is reported. The peptide moiety is composed of two or three repeats of l-Ala-d-Val units while the PEG has a molecular weight of 2kDa. The self-assembling properties of the two conjugates depend significantly on the length of the peptide. Nanoparticles with different sizes and morphologies are formed, the structural properties of which are compared with the previously studied l-Ala-d-Val octapeptide conjugate that self-assembles into rod-like nanoparticles. The aggregation properties were studied by NMR, circular dichroism, fluorescence spectroscopies and dynamic light scattering. The morphology and size of the nanoparticles were assessed by scanning electron microscopy and dynamic light scattering. The loading and release of a model drug were also investigated. This study demonstrates that, by changing the length of the peptide, it is possible to modulate the self-assembly and loading properties of peptide-PEG conjugates.

Novel celecoxib analogues inhibit glial production of prostaglandin E2, nitric oxide, and oxygen radicals reverting the neuroinflammatory responses induced by misfolded prion protein fragment 90-231 or lipopolysaccharide.

We tested the efficacy of novel cyclooxygenase 2 (COX-2) inhibitors in counteracting glia-driven neuroinflammation induced by the amyloidogenic prion protein fragment PrP90-231 or lipopolysaccharide (LPS). In search for molecules with higher efficacy than celecoxib, we focused our study on its 2,3-diaryl-1,3-thiazolidin-4-one analogues. As experimental models, we used the immortalized microglial cell line N9, rat purified microglial primary cultures, and mixed cultures of astrocytes and microglia. Microglia activation in response to PrP90-231 or LPS was characterized by growth arrest, morphology changes and the production of reactive oxygen species (ROS). Moreover, PrP90-231 treatment caused the overexpression of the inducible nitric oxide synthase (iNOS) and COX-2, with the consequent nitric oxide (NO), and prostaglandin E2 (PGE2) accumulation. These effects were challenged by different celecoxib analogues, among which Q22 (3-[4-(sulfamoyl)phenyl]-2-(4-tolyl)thiazolidin-4-one) inhibited microglia activation more efficiently than celecoxib, lowering both iNOS and COX-2 activity and reducing ROS release. During neurodegenerative diseases, neuroinflammation induced by amyloidogenic peptides causes the activation of both astrocytes and microglia with these cell populations mutually regulating each other. Thus the effects of PrP90-231 and LPS were also studied on mixed glial cultures containing astrocytes and microglia. PrP90-231 treatment elicited different responses in the co-cultures induced astrocyte proliferation and microglia growth arrest, resulting in a differential ability to release proinflammatory molecules with the production of NO and ROS mainly attributable on microglia, while COX-2 expression was induced also in astrocytes. Q22 effects on both NO and PGE2 secretion were more significant in the mixed glial cultures than in purified microglia, demonstrating Q22 ability to revert the functional interaction between astrocytes and microglia. These results demonstrate that Q22 is a powerful drug able to revert glial neuroinflammatory responses and might represent a lead to explore the chemical space around celecoxib frameworks to design even more effective agents, paving the way to novel approaches to contrast the neuroinflammation-dependent toxicity.

Asthma Control and Airway Inflammation in Patients with Eosinophilic Granulomatosis with Polyangiitis.

BACKGROUND: Eosinophilic granulomatosis with polyangiitis (EGPA) is a systemic necrotizing vasculitis that occurs in patients with asthma, nasal disease, blood and tissue eosinophilia, and extrapulmonary manifestations. OBJECTIVE: The aim of our study was to assess the clinical, functional, and inflammatory status of upper and lower airways in 37 patients with EGPA, examined 6.4 ± 4.7 years after diagnosis, when they were in partial or complete remission from systemic involvement while on treatment with low-dose oral corticosteroids as maintenance therapy. METHODS: All patients performed spirometry and were assessed for bronchial hyperreactivity, sputum eosinophilia, and fractional exhaled nitric oxide; asthma control was evaluated according to the Global Initiative for Asthma (GINA) guidelines and the Asthma Control Test. Markers of systemic disease were compared with the data available at diagnosis. Nasal involvement was evaluated by using the Sino-Nasal Outcome Test, nasal endoscopy, and nasal cytology. The impact on the quality of life was evaluated by using generic (36-item short form health survey) and organ-specific questionnaires. RESULTS: At the time of the study visit, almost all patients were receiving low-dose oral corticosteroids and immunomodulating drugs, but only 50% were being treated with inhaled corticosteroids. Although low systemic disease activity was documented in the large majority of patients, poorly controlled asthma and rhinosinusitis with eosinophilic airway inflammation were demonstrated in almost all patients. A significant correlation was found between sputum and blood eosinophilia and between fractional exhaled nitric oxide and asthma control. The 36-item short form health survey questionnaire results significantly correlated with the Sino-Nasal Outcome Test but not with the Asthma Control Test. CONCLUSIONS: Systemic treatment controls systemic involvement in EGPA, but not asthma and nasal diseases, which negatively affects patients' quality of life.

Celecoxib Inhibits Prion Protein 90-231-Mediated Pro-inflammatory Responses in Microglial Cells.

Activation of microglia is a central event in the atypical inflammatory response occurring during prion encephalopathies. We report that the prion protein fragment encompassing amino acids 90-231 (PrP90-231), a model of the neurotoxic activity of the pathogenic prion protein (PrP(Sc)), causes activation of both primary microglia cultures and N9 microglial cells in vitro. This effect was characterized by cell proliferation arrest and induction of a secretory phenotype, releasing prostaglandin E2 (PGE2) and nitric oxide (NO). Conditioned medium from PrP90-231-treated microglia induced in vitro cytotoxicity of A1 mesencephalic neurons, supporting the notion that soluble mediators released by activated microglia contributes to the neurodegeneration during prion diseases. The neuroinflammatory role of COX activity, and its potential targeting for anti-prion therapies, was tested measuring the effects of ketoprofen and celecoxib (preferential inhibitors of COX1 and COX2, respectively) on PrP90-231-induced microglial activation. Celecoxib, but not ketoprofen significantly reverted the growth arrest as well as NO and PGE2 secretion induced by PrP90-231, indicating that PrP90-231 pro-inflammatory response in microglia is mainly dependent on COX2 activation. Taken together, these data outline the importance of microglia in the neurotoxicity occurring during prion diseases and highlight the potentiality of COX2-selective inhibitors to revert microglia as adjunctive pharmacological approach to contrast the neuroinflammation-dependent neurotoxicity.

Antiviral activity of benzotriazole derivatives. 5-[4-(Benzotriazol-2-yl)phenoxy]-2,2-dimethylpentanoic acids potently and selectively inhibit Coxsackie Virus B5.

A library of 64 benzotriazole derivatives (17 of which were [4-(benzotriazol-2-yl)phenoxy]alkanoic acids) were screened for antiviral activity against a panel of twelve DNA and RNA viruses. Twenty-six compounds (12 of which were [4-(benzotriazol-2-yl)phenoxy]alkanoic acids) displayed activity against one or more viruses. CVB-5, RSV, BVDV, Sb-1 and YFV were, in decreasing order, the more frequently and effectively affected viruses; DENV-2, WNV, HIV-1 and Reo-1 were only occasionally and modestly affected, while the remaining viruses were not affected by any of the tested compounds. Worth of note were compounds 33 and 35; the former for the activity against Sb-1 (EC50=7 µM) and the latter for the large spectrum of activity including six viruses with a mean EC50=12 µM. Even more interesting were the alkanoic acids 45-48 and 50-57 for their activity against RSV and/or CVB-5. In particular, compound 56 displayed a potent and selective activity against CVB-5 with EC50=0.15 µM and SI=100, thus representing a valuable hit compound for the development of antiviral agents for the treatment of human pathologies related to this virus.

Synthesis and Antiplasmodial Activity of Novel Chloroquine Analogues with Bulky Basic Side Chains.

Chloroquine is commonly used in the treatment and prevention of malaria, but Plasmodium falciparum, the main species responsible for malaria-related deaths, has developed resistance against this drug. Twenty-seven novel chloroquine (CQ) analogues characterized by a side chain terminated with a bulky basic head group, i.e., octahydro-2H-quinolizine and 1,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido[1,2-a][1,5]diazocin-8-one, were synthesized and tested for activity against D-10 (CQ-susceptible) and W-2 (CQ-resistant) strains of P. falciparum. Most compounds were found to be active against both strains with nanomolar or sub-micromolar IC50 values. Eleven compounds were found to be 2.7- to 13.4-fold more potent than CQ against the W-2 strain; among them, four cytisine derivatives appear to be of particular interest, as they combine high potency with low cytotoxicity against two human cell lines (HMEC-1 and HepG2) along with easier synthetic accessibility. Replacement of the 4-NH group with a sulfur bridge maintained antiplasmodial activity at a lower level, but produced an improvement in the resistance factor. These compounds warrant further investigation as potential drugs for use in the fight against malaria.

Multitarget Therapeutic Leads for Alzheimer's Disease: Quinolizidinyl Derivatives of Bi- and Tricyclic Systems as Dual Inhibitors of Cholinesterases and ß-Amyloid (Aß) Aggregation.

Multitarget therapeutic leads for Alzheimer's disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting ß-amyloid (Aß) oligomerization. Thirty-seven thioxanthen-9-one, xanthen-9-one, naphto- and anthraquinone derivatives were tested for the direct inhibition of Aß(1-40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi- and tri-cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of ß-amyloid. In most cases, IC50 values were in the low micromolar and sub-micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC50 =0.84 µM) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and Aß aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics.

Can Sputum Eosinophilia Be a Constant Feature in Severe Refractory Asthmatics? A 3-Year Longitudinal Study.

In difficult-to-treat asthmatics, uncontrolled despite a high level of therapy and followed for 3 years with a mean number of sputum samples/patient = 10, sputum eosinophilia (≥3%) was observed in 87% of all sputum samples. Persistent sputum eosinophilia is a characteristic of severe uncontrolled asthma.