Conformational analysis of a new peptide derived from feline immunodeficiency virus gp36 in SDS micelles: An NMR-MD based investigation.

Feline immunodeficiency virus (FIV) shares structural similarities with human immunodeficiency virus (HIV): the surface glycoprotein gp36 corresponds to the HIV gp41, which drives virus-host cell interactions and is targeted by the peptide entry inhibitor enfuvirtide. Following a similar drug design strategy for the development of an anti-FIV therapy, the present study investigates 627-646gp36 NHR, a peptide sequence derived from a region of gp36 that was previously found to interfere with the antiviral activity of the peptide C8, which instead derives from the gp36 MPER. CD, NMR, and MD simulations were employed to probe the conformational characteristics of 627-646gp36 NHR in the membrane-mimicking environment of SDS micelles. Our data show that 627-646gp36 NHR is characterized by three dynamic helix structures. MD simulations involving 627-646gp36 NHR, C8, and a larger protein, including the CHR and MPER regions, suggest that the interaction of C8 with the MPER region, the origin of the antiviral activity of C8, is disfavored in the presence of 627-646gp36 NHR in the simulation. This evidence can be useful for interpreting the molecular mechanism that leads to interference with the activity of C8, providing information on the folding/unfolding mechanism of the viral glycoprotein to design new strategies to inhibit viral entry.

Monitoring the Conformational Changes of the Aß(25-35) Peptide in SDS Micelles: A Matter of Time.

Alzheimer's disease is a neurodegenerative disease characterized by the formation of amyloid plaques constituted prevalently by amyloid peptides. Due to the well-known challenges related to the study in solution of these peptides, several membrane-mimicking systems such as micelle constituted by detergent-i.e., DPC and SDS-have been deeply investigated. Additionally, the strategy of studying short fragments instead of the full-length peptide turned out to be advantageous in exploring the structural properties of the different moieties in Aß in order to reproduce its pathologic effects. Several studies reveal that among Aß fragments, Aß(25-35) is the shortest fragment able to reproduce the aggregation process. To enrich the structural data currently available, in the present work we decided to evaluate the conformational changes adopted by Aß(25-35) in SDS combining CD and NMR spectroscopies at different times. From the solved structures, it emerges that Aß(25-35) passes from an unordered conformation at the time of the constitution of the system to a more ordered and energetically favorable secondary structure at day 7, which is kept for 2 weeks. These preliminary data suggest that a relatively long time affects the kinetic in the aggregation process of Aß(25-35) in a micellar system, favoring the stabilization and the formation of a soluble helix conformation.

Exploiting the Features of Short Peptides to Recognize Specific Cell Surface Markers.

Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.

Prenatal and Early Postnatal Cerebral d-Aspartate Depletion Influences l-Amino Acid Pathways, Bioenergetic processes, and Developmental Brain Metabolism.

d-Amino acids were believed to occur only in bacteria and invertebrates. Today, it is well known that d-amino acids are also present in mammalian tissues in a considerable amount. In particular, high levels of free d-serine (d-Ser) and d-aspartate (d-Asp) are found in the brain. While the functions of d-Ser are well known, many questions remain unanswered regarding the role of d-Asp in the central nervous system. d-Asp is very abundant at the embryonic stage, while it strongly decreases after birth because of the expression of d-aspartate oxidase (Ddo) enzyme, which catalyzes the oxidation of this d-amino acid into oxaloacetate, ammonium, and hydrogen peroxide. Pharmacologically, d-Asp acts as an endogenous agonist of N-methyl d-aspartate and mGlu5 receptors, which are known to control fundamental brain processes, including brain development, synaptic plasticity, and cognition. In this work, we studied a recently generated knockin mouse model (R26ddo/ddo), which was designed to express DDO beginning at the zygotic stage. This strategy enables d-Asp to be almost eliminated in both prenatal and postnatal lives. To understand which biochemical pathways are affected by depletion of d-Asp, in this study, we carried out a metabolomic and lipidomic study of ddo knockin brains at different stages of embryonic and postnatal development, combining nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) techniques. Our study shows that d-Asp deficiency in the brain influences amino acid pathways such as threonine, glycine, alanine, valine, and glutamate. Interestingly, d-Asp is also correlated with metabolites involved in brain development and functions such as choline, creatine, phosphocholine (PCho), glycerophosphocholine (GPCho), sphingolipids, and glycerophospholipids, as well as metabolites involved in brain energy metabolism, such as GPCho, glucose, and lactate.

Effect of Very-Low-Calorie Ketogenic Diet on Psoriasis Patients: A Nuclear Magnetic Resonance-Based Metabolomic Study.

Psoriasis is an inflammatory disease of the epidermis based on an immunological mechanism involving Langerhans cells and T lymphocytes that produce pro-inflammatory cytokines. Genetic factors, environmental factors, and improper nutrition are considered triggers of the disease. Numerous studies have reported that in a high number of patients, psoriasis is associated with obesity. Excess adipose tissue, typical of obesity, causes a systemic inflammatory status coming from the inflammatory active adipose tissue; therefore, weight reduction is a strategy to fight this pro-inflammatory state. This study aimed to evaluate how a nutritional regimen based on a ketogenic diet influenced the clinical parameters, metabolic profile, and inflammatory state of psoriasis patients. To this end, 30 psoriasis patients were subjected to a ketogenic nutritional regimen and monitored for 4 weeks by evaluating the clinical data, biochemical and clinical parameters, NMR metabolomic profile, and IL-2, IL-1ß, TNF-α, IFN-γ, and IL-4 concentrations before and after the nutritional regimen. Our data show that a low-calorie ketogenic diet can be considered a successful strategy and therapeutic option to gain an improvement in psoriasis-related dysmetabolism, with significant correction of the full metabolic and inflammatory status.

Structural dynamism of chiral sodium peraza-macrocycle complexes derived from cyclic peptoids.

A variety of cyclen and hexacyclen derivatives decorated with (S)-1-phenylethyl side chains or (S)-pyrrolidine units have been prepared via a reductive approach from the corresponding cyclic peptoids containing N-(S)-(1-phenylethyl)glycine and l-proline residues. Spectroscopic and DFT studies on their Na+ complexes show that point chirality and ring size play a crucial role in controlling the structural dynamism of 1,2-diaminoethylene units and pendant arms. The detection of highly symmetric C4- and C3-symmetric metalated species demonstrates that a full understanding of the relationship between the structure and conformational properties of peraza-macrocyclic metal complexes is possible.

Towards an Improvement of Anticancer Activity of Benzyl Adenosine Analogs.

N6-Isopentenyladenosine (i6A) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives' (compounds 2a-m) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2, 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity.

NMR for screening and a biochemical assay: Identification of new FPPS inhibitors exerting anticancer activity.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.

NMR Structure of the FIV gp36 C-Terminal Heptad Repeat and Membrane-Proximal External Region.

Feline immunodeficiency virus (FIV), a lentivirus causing an immunodeficiency syndrome in cats, represents a relevant model of pre-screening therapies for human immunodeficiency virus (HIV). The envelope glycoproteins gp36 in FIV and gp41 in HIV mediate the fusion of the virus with the host cell membrane. They have a common structural framework in the C-terminal region that includes a Trp-rich membrane-proximal external region (MPER) and a C-terminal heptad repeat (CHR). MPER is essential for the correct positioning of gp36 on the lipid membrane, whereas CHR is essential for the stabilization of the low-energy six-helical bundle (6HB) that is necessary for the fusion of the virus envelope with the cell membrane. Conformational data for gp36 are missing, and several aspects of the MPER structure of different lentiviruses are still debated. In the present work, we report the structural investigation of a gp36 construct that includes the MPER and part of the CHR domain (737-786gp36 CHR-MPER). Using 2D and 3D homo and heteronuclear NMR spectra on 15N and 13C double-labelled samples, we solved the NMR structure in micelles composed of dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) 90/10 M: M. The structure of 737-786gp36 CHR-MPER is characterized by a helix-turn-helix motif, with a regular α-helix and a moderately flexible 310 helix, characterizing the CHR and the MPER domains, respectively. The two helices are linked by a flexible loop regulating their orientation at a ~43° angle. We investigated the positioning of 737-786gp36 CHR-MPER on the lipid membrane using spin label-enhanced NMR and ESR spectroscopies. On a different scale, using confocal microscopy imaging, we studied the effect of 737-786gp36 CHR-MPER on 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPC/DOPG) multilamellar vesicles (MLVs). This effect results in membrane budding and tubulation that is reminiscent of a membrane-plasticizing role that is typical of MPER domains during the event in which the virus envelope merges with the host cell membrane.

Effects of Acetylcholine on ß-Amyloid-Induced cPLA2 Activation in the TB Neuroectodermal Cell Line: Implications for the Pathogenesis of Alzheimer's Disease.

The role of ß-amyloid (Aß) in the pathogenesis of Alzheimer's disease (AD) is still considered crucial. The state of Aß aggregation is critical in promoting neuronal loss and neuronal function impairment. Recently, we demonstrated that Acetylcholine (ACh) is neuroprotective against the toxic effects of Aß in the cholinergic LAN-2 cells. In biophysical experiments, ACh promotes the soluble Aß peptide conformation rather than the aggregation-prone ß-sheet conformation. In order to better understand the biological role of ACh in AD, we studied the effect of Aß on the phosphorylation of the cytosolic phospholipase A2 (cPLA2) in the TB neuroectodermal cell line, which differentiates toward a neuronal phenotype when cultured in the presence of retinoic acid (RA). We chose the phosphorylated form of cPLA2 (Ser505, Phospho-cPLA2) as a biomarker to test the influence of ACh on the effects of Aß in both undifferentiated and RA-differentiated TB cells. Our results show that TB cells are responsive to Aß. Moreover, in undifferentiated cells 1 h treatment with Aß induces a 2.5-fold increase of the Phospho-cPLA2 level compared to the control after 24 h in vitro, while no significant difference is observed between Aß-treated and non-treated cells after 4 and 7 days in vitro. The RA-differentiated cells are not sensitive to Aß. In TB cell line ACh is able to blunt the effects of Aß. The ability of ACh to protect non-cholinergic cells against Aß reinforces the hypothesis that, in addition to its role in cholinergic transmission, ACh could also act as a neuroprotective agent.

On the microscopic and mesoscopic perturbations of lipid bilayers upon interaction with the MPER domain of the HIV glycoprotein gp41.

The effect of the 665-683 fragment of the HIV fusion glycoprotein 41, corresponding to the MPER domain of the protein and named gp41MPER, on the microscopic structure and mesoscopic arrangement of palmitoyl oleoyl phosphatidylcholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (CHOL) lipid bilayers is analyzed. The microscopic structuring of the bilayers has been studied by Electron Spin Resonance (ESR) spectroscopy, using glycerophosphocholines spin-labelled in different positions along the acyl chain. Transitions of the bilayer liquid crystalline state have been also monitored by Differential Scanning Calorimetry (DSC). Changes of the bilayers morphology have been studied by determining the dimension of the liposomes through Dynamic Light Scattering (DLS) measurements. The results converge in showing that the sample preparation procedure, the bilayer composition and the peptide/lipid ratio critically tune the lipid response to the peptide/membrane interaction. When gp41MPER is added to preformed liposomes, it positions at the bilayer interface and the lipid perturbation is limited to the more external segments. In contrast, if the peptide is mixed with the lipids during the liposome preparation, it assumes a trans-membrane topology. This happens at all peptide/lipid ratios for fluid POPC bilayers, while in the case of rigid POPC/SM/CHOL membranes a minimum ratio has to be reached, thus suggesting peptide self-aggregation to occur. Peptide insertion results in a dramatic increase of the lipid ordering and bilayer stiffening, which reflect in significant changes in liposome average dimension and distribution. The biological implications of these findings are discussed.

A thermodynamic signature of lipid segregation in biomembranes induced by a short peptide derived from glycoprotein gp36 of feline immunodeficiency virus.

The interactions between proteins/peptides and lipid bilayers are fundamental in a variety of key biological processes, and among these, the membrane fusion process operated by viral glycoproteins is one of the most important, being a fundamental step of the infectious event. In the case of the feline immunodeficiency virus (FIV), a small region of the membrane proximal external region (MPER) of the glycoprotein gp36 has been demonstrated to be necessary for the infection to occur, being able to destabilize the membranes to be fused. In this study, we report a physicochemical characterization of the interaction process between an eight-residue peptide, named C8, modeled on that gp36 region and some biological membrane models (liposomes) by using calorimetric and spectroscopic measurements. CD studies have shown that the peptide conformation changes upon binding to the liposomes. Interestingly, the peptide folds from a disordered structure (in the absence of liposomes) to a more ordered structure with a low but significant helix content. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) results show that C8 binds with high affinity the lipid bilayers and induces a significant perturbation/reorganization of the lipid membrane structure. The type and the extent of such membrane reorganization depend on the membrane composition. These findings provide interesting insights into the role of this short peptide fragment in the mechanism of virus-cell fusion, demonstrating its ability to induce lipid segregation in biomembranes.

Structural features of the C8 antiviral peptide in a membrane-mimicking environment.

C8, a short peptide characterized by three regularly spaced Trp residues, belongs to the membrane-proximal external functional domains of the feline immunodeficiency virus coat protein gp36. It elicits antiviral activity as a result of blocking cell entry and exhibits membranotropic and fusogenic activities. Membrane-proximal external functional domains of virus coat proteins are potential targets in the development of new anti-HIV drugs that overcome the limitations of the current anti-retroviral therapy. In the present work, we studied the conformation of C8 and its interaction with micellar surfaces using circular dichroism, nuclear magnetic resonance and fluorescence spectroscopy. The experimental data were integrated by molecular dynamics simulations in a micelle-water system. Our data provide insight into the environmental conditions related to the presence of the fusogenic peptide C8 on zwitterionic or negatively charged membranes. The membrane charge modulates the conformational features of C8. A zwitterionic membrane surface induces C8 to assume canonical secondary structures, with hydrophobic interactions between the Trp residues and the phospholipid chains of the micelles. A negatively charged membrane surface favors disordered C8 conformations and unspecific superficial interactions, resulting in membrane destabilization.

An Exploratory Bioinformatic Investigation of Cats' Susceptibility to Coronavirus-Deriving Epitopes.

Coronaviruses are highly transmissible and pathogenic viruses for humans and animals. The vast quantity of information collected about SARS-CoV-2 during the pandemic helped to unveil details of the mechanisms behind the infection, which are still largely elusive. Recent research demonstrated that different class I/II human leukocyte antigen (HLA) alleles might define an individual susceptibility to SARS-CoV-2 spreading, contributing to the differences in the distribution of the infection through different populations; additional studies suggested that the homolog of the HLA in cats, the feline leukocyte antigen (FLA), plays a pivotal role in the transmission of viruses. With these premises, this study aimed to exploit a bioinformatic approach for the prediction of the transmissibility potential of two distinct feline coronaviruses (FCoVs) in domestic cats (feline enteric coronavirus (FeCV) and feline infectious peritonitis virus (FIPV)) using SARS-CoV-2 as the reference model. We performed an epitope mapping of nonapeptides deriving from SARS-CoV-2, FeCV, and FIPV glycoproteins and predicted their affinities for different alleles included in the three main loci in class I FLAs (E, H, and K). The predicted complexes with the most promising affinities were then subjected to molecular docking and molecular dynamics simulations to provide insights into the stability and binding energies in the cleft. Results showed the FLA proteins encoded by alleles in the FLA-I H (H*00501 and H*00401) and E (E*01001 and E*00701) loci are largely responsive to several epitopes deriving from replicase and spike proteins of the analyzed coronaviruses. The analysis of the most affine epitope sequences resulting from the prediction can stimulate the development of anti-FCoV immunomodulatory strategies based on peptide drugs.

Investigating the Effectiveness of a Carb-Free Oloproteic Diet in Fibromyalgia Treatment.

Fibromyalgia (FM), a chronic disease with a high incidence in women, poses a significant challenge for diagnosis and treatment, especially due to the absence of specific biomarkers and the multifaceted nature of its symptoms, which range from neuromuscular pain to mood disorders and intestinal dysbiosis. While diagnosis currently relies on rheumatological clinical evaluations and treatment options mainly focus on symptom management, FM seems to have possible links with systemic metabolic dysfunctions with a common inflammatory root. In this context, a new therapeutic avenue emerges: could a therapeutic nutritional approach be the missing piece of the puzzle? Indeed, diet therapies employed particularly for metabolic syndromes proved recently to be efficacious for correcting systemic dysmetabolism and a high number of chronic inflammation conditions. In particular, the very-low-calorie ketogenic diet (VLCKD) demonstrated therapeutic benefits in many disorders. In the present study, we aimed to investigate the specific effects of two dietary interventions, namely the oloproteic VLCKD and the low-glycemic insulinemic (LOGI) diet, on two groups of female FM patients (FM1 and FM2) over a 45-day period. Utilizing clinical and laboratory tests, as well as non-invasive NMR metabolomic analysis of serum, urine, and saliva samples, we sought to uncover how these dietary regimens impact the metabolic dysfunctions associated with FM.

Omega-3 fatty acids regulate the interaction of the Alzheimer's aß(25-35) peptide with lipid membranes.

Polyunsaturated omega-3 fatty acids are increasingly proposed as dietary supplements able to reduce the risk of development or progression of the Alzheimer's disease (AD). To date, the molecular mechanism through which these lipids act has not been yet univocally identified. In this work, we investigate whether omega-3 fatty acids could interfere with the fate of the Alzheimer-related amyloid peptide by tuning the microstructural and dynamical properties of the neuronal membrane. To this aim, the influence of the omega-3 lipid, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine [22:6(cis)PC] on the biophysical properties of lipid bilayers, and on their interaction with the amyloid peptide fragment Aß(25-35) has been investigated by Electron Spin Resonance (ESR), using spin-labeled phospholipids. The results show that the peptide selectively interacts with bilayers enriched in cholesterol (Chol) and sphingomyelin (SM). [22:6(cis)PC] enhances the Aß(25-35)/membrane interaction, favoring a deeper internalization of the peptide among the lipid acyl chains and, consequently, hindering its pathogenic self-aggregation.

Supplementing Low-Sodium Bicarbonate-Calcic (Lete)® Water: Effects in Women on Bone and Systemic Metabolism.

Calcium (Ca) represents about 40% of the total mineral mass, mainly in the bone, providing mechanical strength to the skeleton and teeth. An adequate Ca intake is necessary for bone growth and development in children and adolescents and for maintaining bone mineral loss in elderly age. Ca deficiency predisposes to osteopenia and osteoporosis. Healthy nutrition, including an adequate intake of Ca-rich food, is paramount to prevent and cure osteoporosis. Recently, several clinical studies have demonstrated that, in conditions of Ca dysmetabolism, Ca-rich mineral water is beneficial as a valuable source of Ca to be used as an alternative to caloric Ca-rich dairy products. Although promising, these data have been collected from small groups of participants. Moreover, they mainly regard the effect of Ca-rich mineral water on bone metabolism. In contrast, an investigation of the effect of Ca supplementation on systemic metabolism is needed to address the spreading of systemic metabolic dysfunction often associated with Ca dysmetabolism. In the present study, we analyzed urine and blood sera of 120 women in perimenopausal condition who were subjected for six months to 2l daily consumption of bicarbonate-calcium mineral water marketed under ®Lete. Remarkably, this water, in addition to being rich in calcium and bicarbonate, is also low in sodium. A complete set of laboratory tests was carried out to investigate whether the specific water composition was such to confirm the known therapeutic effects on bone metabolism. Second, but not least, urine and blood sera were analyzed using NMR-based metabolomic procedures to investigate, other than the action on Ca metabolism, potential system-wide metabolic effects. Our data show that Lete water is a valid supplement for compensating for Ca dysmetabolism and preserving bone health and integrity.

SMN deficiency perturbs monoamine neurotransmitter metabolism in spinal muscular atrophy.

Beyond motor neuron degeneration, homozygous mutations in the survival motor neuron 1 (SMN1) gene cause multiorgan and metabolic defects in patients with spinal muscular atrophy (SMA). However, the precise biochemical features of these alterations and the age of onset in the brain and peripheral organs remain unclear. Using untargeted NMR-based metabolomics in SMA mice, we identify cerebral and hepatic abnormalities related to energy homeostasis pathways and amino acid metabolism, emerging already at postnatal day 3 (P3) in the liver. Through HPLC, we find that SMN deficiency induces a drop in cerebral norepinephrine levels in overt symptomatic SMA mice at P11, affecting the mRNA and protein expression of key genes regulating monoamine metabolism, including aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DßH) and monoamine oxidase A (MAO-A). In support of the translational value of our preclinical observations, we also discovered that SMN upregulation increases cerebrospinal fluid norepinephrine concentration in Nusinersen-treated SMA1 patients. Our findings highlight a previously unrecognized harmful influence of low SMN levels on the expression of critical enzymes involved in monoamine metabolism, suggesting that SMN-inducing therapies may modulate catecholamine neurotransmission. These results may also be relevant for setting therapeutic approaches to counteract peripheral metabolic defects in SMA.

The iAß5p ß-breaker peptide regulates the Aß(25-35) interaction with lipid bilayers through a cholesterol-mediated mechanism.

Alzheimer's disease is characterized by the deposition of aggregates of the ß-amyloid peptide (Aß) in the brain. A potential therapeutic strategy for Alzheimer's disease is the use of synthetic ß-sheet breaker peptides, which are capable of binding Aß but unable to become part of a ß-sheet structure, thus inhibiting the peptide aggregation. Many studies suggest that membranes play a key role in the Aß aggregation; consequently, it is strategic to investigate the interplay between ß-sheet breaker peptides and Aß in the presence of lipid bilayers. In this work, we focused on the effect of the ß-sheet breaker peptide acetyl-LPFFD-amide, iAß5p, on the interaction of the Aß(25-35) fragment with lipid membranes, studied by Electron Spin Resonance spectroscopy, using spin-labeled membrane components (either phospholipids or cholesterol). The ESR results show that iAß5p influences the Aß(25-35) interaction with the bilayer through a cholesterol-mediated mechanism: iAß5p withholds cholesterol in the inner hydrophobic core of the bilayer, making the interfacial region more fluid and capable to accommodate Aß(25-35). As a consequence, iAß5p prevents the Aß(25-35) release from the lipid membrane, which is the first step of the ß-amyloid aggregation process.

Development and application of a fast ultra-high performance liquid chromatography-trapped ion mobility mass spectrometry method for untargeted lipidomics.

The alteration of lipid profile in biological specimens, such as plasma, mirrors abnormalities in their homeostasis and offers pivotal information for disease comprehension. Fast analytical methods are needed to highlight changes in plasma lipid profile and deliver rapid results. In this study we developed a fast reversed phase ultra high performance liquid chromatography-trapped ion mobility mass spectrometry (RP-UHPLC-TIMS-MS) method for untargeted lipidomics. A short, narrow-bore fully porous particle CSH column (50 mm × 2.1 mm, 1.7 µm) was used, and by selecting appropriate flow rate, temperature and gradient conditions, the total analysis time was reduced from 20 to 4 min. TIMS was operated in parallel accumulation serial fragmentation mode (PASEF) which allowed to select multiple precursors for MS/MS and separate co-eluting lipids based on their different mobility. Lipid annotation was performed by rule-based approach, comparison with LipidBlast spectral library and manual data curation, by taking into account class-specific fragmentation pattern, accurate mass, adduct form, retention behavior in RP and comparison of their collision cross-section (CCS) values for increased confidence. 306 unique lipids from 21 subclasses were annotated from 20 µL of plasma, while their concentration was estimated by class-specific deuterated internal standards. The analytical method was validated and finally applied to elucidate the alteration of plasma lipid profiles in a small cohort of amyotrophic lateral sclerosis (ALS) patients. Univariate and multivariate statistics evidenced significant differences with respect to control patients, particularly in the levels of ether linked lipids (PC-O, PE-O, PE-P and LPC-O), sphingolipids (Ceramides), and triacylglycerols, showing the usefulness of this fast approach in providing accurate and rapid results with respect to longer (≥15 min) untargeted UHPLC-HRMS methods.