Sequencing Biodegradable and Potentially Biobased Polyesteramide of Sebacic Acid and 3-Amino-1-propanol by MALDI TOF-TOF Tandem Mass Spectrometry.

Biodegradable and potentially biobased polyesteramide oligomers (PEA-Pro), obtained from melt condensation of sebacic acid and 3-amino-1-propanol, were characterized by nuclear magnetic resonance (NMR), matrix assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry/mass spectrometry (MALDI-TOF/TOF-MS/MS), thermogravimetric analysis (TGA), and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). NMR analysis showed the presence of hydroxyl and amino terminal groups as well as carboxylic groups of the sebacate moiety. Hydroxyl and carboxyl termination had the same abundance, while the amine termination was 2.7-times less frequent. Information regarding the fragmentation pathways and ester/amide bond sequences was obtained by MALDI-TOF/TOF-MS/MS analysis performed on sodiated adducts of cyclic species and linear oligomers. Different end groups did not influence the observed fragmentation. Three fragmentation pathways were recognized. The ß-hydrogen-transfer rearrangement, which leads to the selective scission of the -O-CH2- bonds, was the main mechanism. Abundant product ions originating from -CH2-CH2- (ß-γ) bond cleavage in the sebacate moiety and less abundant ions formed by -O-CO- cleavages were also detected. TGA showed a major weight loss (74%) at 381 °C and a second degradation step (22% weight loss) at 447 °C. Py-GC/MS performed in the temperature range of 350-400 °C displayed partial similarity between the degradation products and the main fragments detected in the MALDI-TOF/TOF-MS/MS experiments. Degradation products derived from amide bonds were related to the formation of CN groups, in agreement with the literature.

Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients.

Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.

Switch of predicted HIV-1 tropism in treated subjects and its association with disease progression.

Dynamics of human immunodeficiency virus type 1 (HIV-1) tropism after antiretroviral therapy (ART) initiation and their association with disease progression are poorly investigated.This was a cohort study on subjects from the ICONA cohort receiving ART with persistently detectable (PD) or persistently undetectable (PU) viral load (VL) and with stored plasma or peripheral blood mononuclear cell (PBMC) samples at 2 time-points (T1, T2) after ART initiation. HIV-1 co-receptor tropism was determined by V3-loop sequencing and the geno2pheno algorithm. A switch in viral tropism was defined if the tropism classification at T2 differed from that observed at T1. Time to disease progression, defined as the occurrence of a new acquired immune deficiency syndrome (AIDS)-defining event/death from T2, was also evaluated.One hundred ninety-five patients were analyzed (124 PD, 71 PU). Over a median follow-up of 22.6 (19.8-28.1) months, PD and PU patients showed similar rates (95% confidence interval) of switch to a non-R5 virus [PD: 6.9 (3.7-11.2)/100-person-years of follow-up (PYFU); PU: 8.0 (3.4-14.5)/100-PYFU; P = 0.63] and of switch to a R5 virus [PD: 15.4 (7.3-26.4)/100-PYFU; PU: 8.1 (2.5-16.7)/100-PYFU; P = 0.38]. Switch to non-R5 virus was predicted by nadir CD4+ before T1.Twenty-two (18%) PD and 4 (6%) PU subjects experienced disease progression (P = 0.02). The risk of disease progression was independently associated with a switch in co-receptor tropism (adjusted hazard ratio = 4.06, 95% CI: 1.20-13.80, P = 0.03) as well as age, AIDS diagnosis, nadir CD4+ before T2, current CD4+, and VL.Switch of HIV-1 tropism under ART occurs in both directions, with similar rates in subjects with PD or PU VL and it might be predictive of future unfavorable clinical outcome.

The HIV-1 reverse transcriptase polymorphism A98S improves the response to tenofovir disoproxil fumarate+emtricitabine-containing HAART both in vivo and in vitro.

The impact of baseline HIV-1 reverse transcriptase (RT) polymorphisms on response to first-line modern HAART containing tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) was evaluated. The impact of each RT polymorphism on virological success (VS) was evaluated in 604 HIV-1 subtype B-infected patients starting TDF+FTC-containing HAART. TDF and FTC antiviral activity was also tested in PBMCs infected by mutagenised HIV. Structural analysis based on docking simulations was performed. A98S was the only mutation significantly correlated with an increased proportion of patients achieving VS at 24 weeks (94.0% vs. 84.3%; P=0.03). Multivariate regression and Cox model analyses confirmed this result. At concentrations close to the minimal concentration achieved in patient plasma, TDF and FTC exhibited higher potency in the presence of A98S-mutated virus compared with wild-type (IC90,TDF, 8.6±1.1 vs. 19.3±3.5nM; and IC90,FTC, 12.4±7.7 vs. 16.8±9.8nM, respectively). The efficacy of FTC, abrogated by M184V, was partially restored by A98S (IC90,FTC, 5169±5931nM for A98S+M184V vs. 18477±12478nM for M184V alone). Docking analysis showed the higher potency of TDF and FTC in the presence of A98S-mutated virus was mainly due to higher binding affinity between drugs and mutated RT compared with wild-type. In the presence of FTC, A98S also partially restored the RT binding affinity impaired by M184V alone. A98S polymorphism improves virological response to TDF+FTC-containing HAART. This may help clinicians in the choice of the optimal NRTI backbone aimed at achieving maximal virological inhibition.

Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage.

Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e-7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, -40.1 kcal/mol; G24E, -510 kcal/mol; E25K, -522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression.

Prostaglandin A1 inhibits avian influenza virus replication at a postentry level: Effect on virus protein synthesis and NF-κB activity.

Influenza A viruses (IAV) have the potential to cause devastating pandemics. In recent years, the emergence of new avian strains able to infect humans represents a serious threat to global human health. The increase in drug-resistant IAV strains underscores the need for novel approaches to anti-influenza chemotherapy. Herein we show that prostaglandin-A1 (PGA1) possesses antiviral activity against avian IAV, including H5N9, H7N1 and H1N1 strains, acting at a level different from the currently available anti-influenza drugs. PGA1 acts at postentry level, causing dysregulation of viral protein synthesis and preventing virus-induced disassembly of host microtubular network and activation of pro-inflammatory factor NF-κB. The antiviral activity is dependent on the presence of a cyclopentenone ring structure and is associated with activation of a cytoprotective heat shock response in infected cells. The results suggest that cyclopentenone prostanoids or prostanoids-derived molecules may represent a new tool to combat avian influenza virus infection.

Reliability and clinical relevance of the HIV-1 drug resistance test in patients with low viremia levels.

BACKGROUND: We evaluated reliability and clinical usefulness of genotypic resistance testing (GRT) in patients for whom combination antiretroviral therapy (cART) was unsuccessful with viremia levels 50-1000 copies/mL, for whom GRT is generally not recommended by current guidelines. METHODS: The genotyping success rate was evaluated in 12 828 human immunodeficiency virus type 1 (HIV-1) plasma samples with viremia >50 copies/mL, tested using the commercial ViroSeq HIV-1 Genotyping System or a homemade system. Phylogenetic analysis was performed to test the reliability and reproducibility of the GRT at low-level viremia (LLV). Drug resistance was evaluated in 3895 samples from 2200 patients for whom treatment was unsuccessful (viremia >50 copies/mL) by considering the resistance mutations paneled in the 2013 International Antiviral Society list. RESULTS: Overall, the success rate of amplification/sequencing was 96.4%. Viremia levels of 50-200 and 201-500 copies/mL afforded success rates of 67.2% and 88.1%, respectively, reaching 93.2% at 501-1000 copies/mL and ≥97.3% above 1000 copies/mL. A high homology among sequences belonging to the same subject for 96.4% of patients analyzed was found. The overall resistance prevalence was 74%. Drug resistance was commonly found also at LLV. In particular, by stratifying for different viremia ranges, detection of resistance was as follows: 50-200 copies/mL = 52.8%; 201-500 = 70%; 501-1000 = 74%; 1001-10 000 = 86.1%; 10 001-100 000 = 76.7%; and >100 000 = 63% (P < .001). Similar bell-shaped results were found when the GRT analysis was restricted to 2008-2012, although at a slightly lower prevalence. CONCLUSIONS: In patients failing cART with LLV, HIV-1 genotyping provides reliable and reproducible results that are informative about emerging drug resistance.

Sexual dimorphic evolution of metabolic programming in non-genetic non-alimentary mild metabolic syndrome model in mice depends on feed-back mechanisms integrity for pro-opiomelanocortin-derived endogenous substances.

Previously, we showed that our post-natal handling model induces pro-opiomelanocortin-derived (POMC) endogenous systems alterations in male mice at weaning. These alterations last up to adult age, and are at the basis of adult hormonal and metabolic conditions similar to mild metabolic syndrome/type-2 diabetes. Here, we evaluate how sex influences post-natal programming in these metabolic conditions. Subjects are adult control (non-handled) female (NHF) and male (NHM) CD-1 mice; adult post-natal handled female (HF) and male (HM) mice. Handling consists of daily maternal separation (10 min) plus sham injection, from birth to weaning (21 days). In adult handled males (90-days old) we find not only POMC-derived hormones alterations (enhanced basal plasma corticosterone (+91%) and ACTH (+109%)) but also overweight (+5.4%), fasting hyperglycemia (+40%), hypertriglyceridemia (+21%), enhanced brain mRNA expression of hydroxysteroid(11-beta)dehydrogenase type-1 (HSD11B1) (+49%), and decreased mRNA-HSD11B2 (-39%). Conversely, uric acid, creatinine, HDL(C), total cholesterol, glucose and insulin incremental area under-the-curve are not affected. In females, post-natal handling does not produce both hormonal and dysmetabolic diabetes-like changes; but handling enhances n3- and n6-poly-unsaturated, and decreases saturated fatty acids content in erythrocyte membrane composition in HF versus NHF. In conclusion, for the first time we show that female sex in mice exerts effective protection against the hypothalamus-pituitary-adrenal homeostasis disruption induced by our post-natal handling model on POMC cleavage products; endocrine disruption is in turn responsible for altered metabolic programming in male mice. The role of sex hormones is still to be elucidated.

Neonatal taurine administration modifies metabolic programming in male mice.

The semi-essential amino-acid taurine is involved in glucose homeostasis either in adults or in parental life. Taurine is currently used in neonatal life because it is added to milk formula for babies, and to parental solution for prematures. Here, it has been examined whether taurine administration in lactation modifies adult glucose metabolism. Neonatally taurine-treated mice (50 mg/kg body weight/day, for the first 21 days of life) as adults have lower basal glucose and iAUC after glucose loading curves in comparison with vehicle-treated mice, whereas iAUC following insulin loading curves, plasma lipids and malondialdehyde (MDA), an index of lipid peroxidation were not significantly changed. Thus, in rodents, neonatally administered taurine produces enduring effects in a way that could be advantageous for the control of glucose homoeostasis.

Antioxidant effect of two virgin olive oils depends on the concentration and composition of minor polar compounds.

In vitro studies show that some individual minor polar phenolic compounds (MPC) present in virgin olive oil prevent oxidation of human low-density lipoproteins (LDL), but few data are available on the antioxidant effect of whole oil extract. Thus, whole virgin olive extracts were studied to determine whether they maintain the antioxidant activity and whether this last is linked to MPC composition of a single virgin oil. Using HPLC-DAD the MPC content in Taggiasca and Seggianese virgin olive oils was measured. Taggiasca oil was less rich in total MPC (208.5 mg/L) than Seggianese oil (441.9 mg/L). In addition, the major compounds of Taggiasca oil were lignan derivatives, whereas the major compounds in Seggianese oils were secoiridoid derivatives. Moreover, Taggiasca oil was practically free of 5-hydroxytyrosol and 5-hydroxytyrosol derivatives, deacetoxy-oleuropein aglycone and oleuropein aglycone. The antioxidant activity of the oils on human LDL was evaluated by measuring malondialdehyde and conjugate diene generation induced by copper ions. In both tests, the oil extracts dose-dependently reduced malondialdehyde and conjugate diene generation. Moreover, antioxidant potency correlated with total MPC; thus, Seggianese extract was more active. The two oils differed quantitatively and qualitatively, and these differences influenced their biological activities; thus clinical trials focused on studying the effects of olive oils should specify the oils used.

Evaluation of antioxidant effect of different extracts of Myrtus communis L.

Oxidative stress is involved in the pathogenesis of numerous diseases. Nevertheless, no optimal natural antioxidant has been found for therapeutics, therefore polyphenol antioxidants have been looked for in myrtle leaves, a plant that in folk medicine has been used as anti-inflammatory drug. Antioxidant-rich fractions were prepared from myrtle (Myrtus communis L.) leaves liquid-liquid extraction (LLE) with different solvents. All myrtle extracts were very rich in polyphenols. In particular, hydroalcoholic extracts contain galloyl-glucosides, ellagitannins, galloyl-quinic acids and flavonol glycosides; ethylacetate extract and aqueous residues after LLE are enriched in flavonol glycosides and hydrolysable tannins (galloyl-glucosides, ellagitannins, galloyl-quinic acids), respectively. Qualitative and quantitative analysis for the single unidentified compound was also performed. Human LDL exposed to copper ions was used to evaluate the antioxidant activity of the myrtle extracts. Addition of these extracts did not affect the basal oxidation of LDL but dose-dependently decreased the oxidation induced by copper ions. Moreover, the myrtle extracts reduce the formation of conjugated dienes. The antioxidant effect of three myrtle extracts decreased in the following order: hydroalcoholic extracts, ethylacetate and aqueous residues after LLE. The extracts had the following IC50: 0.36, 2.27 and 2.88 microM, when the sum of total phenolic compounds was considered after the correction of molecular weight based on pure compounds. Statistical analysis showed a significant difference among hydroalcoholic extracts vs. the ethylacetate and aqueous residues after LLE. These results suggest that the myrtle extracts have a potent antioxidant activity mainly due to the presence of galloyl derivatives.