Sensitivity of serology assay in Covid-19 diagnosis: does the antigen matter?

Since the spreading of Sar-CoV-2 in March 2020, many serologic tests have been developed to identify antibody responses. Indeed, different commercial kits are directed against different antigens and could utilise different methods thereby triggering confusion and criticism. Here, we compared two Food and Drug Administration (FDA)-approved automatized assays that detect IgG responses against spike or nucleocapsid protein of Sars-Cov-2 virus in 127 subjects among healthcare workers of IRCCS Policlinico San Donato (MI), Italy. We observed different kinetics of IgG responses, demonstrating the importance of timing of sampling to correctly interpret the results both for infection diagnosis and for epidemiologic studies. We observed that Anti-N response starts earlier than Anti-S1/S2 response but also decreases earlier, affecting the sensitivity of the tests at different time points. Combining two different assays, designed against different antigens, could reduce false negative results. Finally, we observed a patient who produced anti-nucleocapsid IgG, but not anti-spike IgG. In conclusion, we investigated antibody responses in Covid-19 disease, aiming to direct clinicians and laboratory scientists to correctly interpret serologic results by always paying attention to clinical history correlation, timing of sampling, methods and antigens used, to avoid false negative results and obtain relevant epidemiologic data.

Temporal dynamics in the evolution of the sunflower genome as revealed by sequencing and annotation of three large genomic regions.

Improved knowledge of genome composition, especially of its repetitive component, generates important informations in both theoretical and applied research. In this study, we provide the first insight into the local organization of the sunflower genome by sequencing and annotating 349,380 bp from 3 BAC clones, each including one single-copy gene. These analyses resulted in the identification of 11 putative gene sequences, 18 full-length LTR retrotransposons, 6 incomplete LTR retrotransposons, 2 non-autonomous LTR-retroelements (LINEs), 2 putative DNA transposons fragments and one putative helitron. Among LTR-retrotransposons, non-autonomous elements (the so-called LARDs), which do not carry any protein-encoding sequence, were discovered for the first time in the sunflower. The insertion time of intact retroelements was measured, based on sister LTRs divergence. All isolated elements were inserted relatively recently, especially those belonging to the Gypsy superfamily. Retrotransposon families related to those identified in the BAC clones are present also in other species of Helianthus, both annual and perennial, and even in other Asteraceae. In one of the three BAC clones, we found five copies of a lipid transfer protein (LTP) encoding gene within less than 100,000 bp, four of which are potentially functional. Two of these are interrupted by LTR retrotransposons, in the intron and in the coding sequence, respectively. The divergence between sister LTRs of the retrotransposons inserted within the genes indicates that LTP gene duplication started earlier than 1.749 MYRS ago. On the whole, the results reported in this study confirm that the sunflower is an excellent system to study transposons dynamics and evolution.

HACRE1, a recently inserted copia-like retrotransposon of sunflower (Helianthus annuus L.).

In this paper we report on the isolation and characterization, for the first time, of a complete 6511 bp retrotransposon of sunflower. Considering its protein domain order and sequence similarity to other copia elements of dicotyledons, this retrotransposon was assigned to the copia retrotransposon superfamily and named HACRE1 (Helianthus annuus copia-like retroelement 1). HACRE1 carries 5' and 3' long terminal repeats (LTRs) flanking an internal region of 4661 bp. The LTRs are identical in their sequence except for two deletions of 7 and 5 nucleotides in the 5' LTR. Based on the sequence identity of the LTRs, HACRE1 was estimated to have inserted within the last approximately 84 000 years. The isolated sequence contains a complete open reading frame with only one complete reading frame. The absence of nonsense mutations agrees with the very high sequence identity between LTRs, confirming that HACRE1 insertion is recent. The haploid genome of sunflower (inbred line HCM) contains about 160 copies of HACRE1. This retrotransposon is expressed in leaflets from 7-day-old plantlets under different light conditions, probably in relation to the occurrence of many putative light-related regulatory cis-elements in the LTRs. However, sequenced cDNAs show less variability than HACRE1 genomic sequences, indicating that only a subset of this family is expressed under these conditions.

Salvia somalensis essential oil as a potential cosmetic ingredient: solvent-free microwave extraction, hydrodistillation, GC-MS analysis, odour evaluation and in vitro cytotoxicity assays.

Salvia somalensis Vatke, a wild sage native of Somalia, has been studied with the aim of assessing the potential cosmetic application of its essential oil, recovered from fresh aerial parts by solvent-free microwave extraction - SFME. To evaluate the efficiency and reliability of this eco-friendly procedure, the recovery of the essential oil was also processed by conventional hydrodistillation (HD) and the results compared. The essential oils obtained by both SFME and HD were analysed by gas chromatography-mass spectrometry using apolar and polar capillary columns. The essential oil recovered by SFME was submitted to an odour evaluation that revealed peculiar olfactive characteristics interesting in alcoholic male perfumery and body detergents.In vitro cytotoxicity assays were carried out using NCTC 2544 human keratinocytes as target cells. The oil displayed slight cytotoxic effects, which were three orders of magnitude lower than those found for sodium dodecyl sulphate positive control. The promising results in terms of chemical composition, scent and safety seem to indicate this essential oil as an interesting potential functional ingredient useful in a cosmetic context.

Estradiol-antagonistic activity of phenolic compounds from leguminous plants.

Natural flavonoids are currently receiving much attention because of their estrogenic and antiestrogenic properties. Six isoflavones (isoprunetin, isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside, genistein, genistein 7-O-beta-D-glucopyranoside, daidzein), four flavones (luteolin, luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C), isolated from Genista morisii and G. ephedroides (two Leguminosae plants of the Mediterranean area) together with two structurally related pterocarpans, bitucarpin A and erybraedyn C, isolated from Bituminaria bituminosa (Leguminosae), were tested for the antagonist activity by a yeast based estrogen receptor assay (Saccharomyces cerevisiae RMY326 ER-ERE). Most compounds inhibited the estradiol-induced transcriptional activity in a concentration dependent manner. In particular, for the flavone luteolin 77% inhibition of the induced beta-galactosidase activity was observed. Interestingly, licoflavone C exhibited a dose-dependent antagonistic activity at concentrations up to 10(-4) M, but stimulated beta-galactosidase expression at higher concentrations resulting in a U-shaped-like dose-response curve.

Essential oils in medicine: principles of therapy.

Essential oils (EOs) are extremely complex mixtures containing compounds of several different functional-group classes. A specific aromatic profile should be determined by gas-chromatography-mass detection methods, to define standards for their safety and efficacy. The chemical constituents of the essential oils, their flavour and their taste act both alone and in synergy, always determining a global psychosomatic action. The main therapeutic activities of the EOs are reported as spasmolythic, revulsive, anti-inflammatory and decongestant, immunomodulant, antimicrobial, antimycotic, expectorant, mucolythic, antioxidant, psychotrope, analgesic and acaricide. The use, posology, route of administration as well as toxicity and adverse effects are reviewed.

Arbuscular mycorrhizal fungi alter the content and composition of secondary metabolites in Bituminaria bituminosa L.

Secondary metabolites may be affected by arbuscular mycorrhizal fungi (AMF), which are beneficial symbionts associated with the roots of most plant species. Bituminaria bituminosa (L.) C.H.Stirt is known as a source of several phytochemicals and therefore used in folk medicine as a vulnerary, cicatrising, disinfectant agent. Characteristic metabolites found in B. bituminosa are furanocoumarins and pterocarpans, which are used in cosmetics and as chemotherapeutic agents. Here we address the question whether AMF inoculation might affect positively the synthesis of these phytochemicals. B. bituminosa plants were inoculated with different AMF and several metabolites were assessed during full vegetative stage and flowering phase. Pigments (chlorophylls and carotenoids), polyphenols and flavonoids were spectrophotometrically determined; specific isoflavones (genistein), furanocoumarins (psoralene and angelicin), pterocarpans (bitucarpin A and erybraedin C) and plicatin B were assessed with HPLC; leaf volatile organic compounds were analysed using SPME and identified by GC-MS. During the vegetative stage, the inoculated plants had a high amount of furanocoumarins (angelicin and psoralen) and pterocarpans (erybraedin C and bitucarpin A). The analysis of volatile organic compounds of inoculated plants showed different chemical composition compared with non-mycorrhizal plants. Given the important potential role played by furanocoumarins and pterocarpans in the pharmaceutical industry, AMF inoculation of B. bituminosa plants may represent a suitable biotechnological tool to obtain higher amounts of such metabolites for pharmaceutical and medicinal purposes.

Phytochemical study of Bituminaria basaltica aerial parts, an Italian endemism.

The first phytochemical investigation of the aerial parts of Bituminaria basaltica, an endemic species from the Aeolian Islands, led to the isolation and identification of eight compounds including plicatin B (3), two furanocoumarins: angelicin (1), psoralen (2), three pterocarpans: erybraedin C (4), 3,9-dihydroxy-4-isoprenyl-pterocarpan (5), bitucarpin A (8) and two flavonoid glycosides: isoorientin (6), daidzin (7). Their structures were elucidated by spectroscospic techniques and compared with data reported in the literature. Sesquiterpenes characterised the essential oil composition of the title plant where ß-caryophyllene and germacrene D were the main constituents.

Minor constituents from Bupleurum fruticosum roots.

A new triterpenoid saponin, 23-acetoxy,16 alpha-hydroxy-13,28-epoxyolean-11-en-3 beta-yl--beta-D-glucopyranosyl- (1-->2)---beta-D-glucopyranosil(1-->3)-beta-D-fucopyranoside, has been isolated from the roots of Bupleurum fruticosum, along with eight coumarins, scopoletin, scoparone, prenyletin, capensin, fraxetin, aesculetin,7-(3-methyl-2-butenyloxy-6-methoxycoumarin, 7-(2-hydroxy-3-methyl-3-butenyloxy)-6-methoxycoumarin and 5,7-dihydroxy-6-methoxy-8-(3-methyl-2-butenyl)coumarin. The last compound has not been previously encountered in nature. The structures were determined by analysis of NMR spectral data including two-dimensional techniques.

Flavonoids from genista ephedroides

Three new flavonoids (1-3) and 11 known compounds (genistein, isoprunetin, wighteone, laburnetin, alpinumisoflavone, genistin, genistein 8-C-glucoside, apigenin, isokaempferide, licoflavone C, and D-pinitol) were isolated from the aerial parts of Genista ephedroides. The structures of the new compounds were established as hydroxyalpinumisoflavone (1) [4', 5-dihydroxy-2"-methyl-2"-hydroxymethylpyrano(5",6":6,7)isoflavone], ephedroidin (2) [4',5, 7-trihydroxy-8-(2-hydroxy-3-methyl-3-butenyl)flavone], and genisteone (3) (7-O-glucosylwighteone) by means of spectroscopic methods.

Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes.

Plant-derived isoflavones are currently receiving much attention because of their phyto-estrogenic, antioxidant, anti-mutagenic, and anti-tumor activities. In this study we have evaluated the clastogenic and anti-clastogenic activities in human lymphocytes of two structurally related pterocarpans, iso-flavonoid derivatives, termed erybraedin C and bitucarpin A, recently purified from Bituminaria bituminosa and chemically characterized. Mitomycin C (MMC) and the radio-mimetic bleomycin (BL) were used as reference clastogens. The end point studied was micronucleus formation. The results obtained in this study indicate that erybraedin C and bitucarpin A, when assayed alone, do not affect either the mitotic index or the cell-proliferation index of human lymphocytes. Interestingly, both compounds appear to be non-clastogenic in the range of concentrations used. In contrast, both substances seem to affect significantly the clastogenic effects induced by BL and MMC. A 1-h pre-exposition of the cell culture to erybraedin C was necessary to display its anti-clastogenic potential against BL, whereas bitucarpin A was inactive in this respect, with a structure-activity relationship. In contrast, the clastogenic activity of MMC was significantly reduced by both erybraedin C and bitucarpin A, using either a pre-incubation schedule or simultaneous treatment. These results suggest that the protective effects displayed by the two anti-clastogenic compounds against MMC could be due to the induction or inhibition of cellular reductive metabolic enzymes.

Induction of binucleation in human lymphocytes by 14 synthetic isoindolone derivatives related to cytochalasins.

Fourteen hydrogenated isoindolone derivatives with affinity to cytochalasin B were synthesized and tested for the induction of binucleation in the human lymphocyte micronucleus (MN) assay. The experimental procedure was the one commonly used for the human lymphocyte MN assay. Compounds like cyt-B were added to cultures at 44 h and tested at increasing concentrations, up to 200 mumol/l, in the range commonly used for cyt-B in the MN assay (3 micrograms/ml = 6.25 mumol/l). Induction of cytokinesis-blocked binucleated cells was found for all compounds but, at the same molarity, cyt-B induced a higher percentage of binucleation. Only one of the compounds tested was found to induce micronuclei significantly: MN were induced across the dose range of 25-100 mumol/l.

Different effects of newly isolated saponins on the mutagenicity and cytotoxicity of the anticancer drugs mitomycin C and bleomycin in human lymphocytes.

Aim of the present paper was to assess by using the in vitro micronucleus (MN) test in human lymphocytes the effect of two plant extracts isolated from Blupeurum fruticosum (saponins) on the clastogenicity and cytotoxicity of the anticancer drugs mitomycin C (MMC) and bleomycin (BLM). One saponin showed a dose-dependent MMC-induced mutagenesis inhibition together with co-genotoxic effect on BLM-treated cultures. The remaining saponin did not significantly alter MN induction of both chemotherapeutic agents whereas it enhanced BLM cytotoxicity.

In vitro genotoxicity and cytotoxicity of five new chemical compounds of plant origin by means of the human lymphocyte micronucleus assay.

The micronucleus test in human peripheral lymphocytes is widely used in toxicology for the assessment of the genotoxic profile of chemical compounds of environmental concern. The aim of this study was to evaluate the genotoxic, cytotoxic and antimitotic activity of five new compounds isolated from Prunus africana Hook or from Bupleurum fruticosum L. The experiments were conducted only in vitro. Results showed that none of the plant extracts, tested over a wide range of concentrations, increased the frequency of micronuclei. Only compounds 2 and 5 were found to be toxic for phytohaemagglutinin-stimulated lymphocytes at the maximum dose used. reserved.

Synthesis and biological activity of a series of isoindolone derivatives related to cytochalasins.

A series of hydrogenated isoindolone derivatives structurally related to cytochalasin B has been synthesized and their ability to induce binucleation in the human lymphocytes were tested. All compounds were found able to inhibit cell cytokinesis at different extent in the range (3.12-25 mumol/l) respect to the negative control; however the highest percentage of binucleated cells is induced by Cyt B.

Flavonoids from Calicotome villosa.

Two new acylated flavone glycosides, chrysin 7-(6"-O-acetyl)-O-beta-D-glucopyranoside (1) and chrysin 7-(4"-O-acetyl)-O-beta-D-glucopyranoside (2) were isolated from the aerial parts of Calicotome villosa, along with 17 known flavonoids and one triterpene.

Antimicrobial activity of crude extracts and pure compounds of Hypericum hircinum.

The antimicrobial activity of the n-hexane (H) and chloroform (C) extracts, the methanol extract (M) and its ethyl acetate (E) and n-butanol (B) fractions, and six isolated constituents of the aerial parts of Hypericum hircinum was investigated using an agar diffusion method. The maximum activity was exhibited by the methanolic extract against Staphylococcus aureus, while all pure constituents showed no antimicrobial activity against the tested microorganisms.

Quinolizidine alkaloids from Genista ephedroides.

Alkaloids retamine, anagyrine, lupanine, 17-oxoretamine, 12-alpha-hydroxylupanine were detected, along with four others unidentified compounds in the aerial parts of Genista ephedroides D.C.

Clinical and mycological evaluation of an herbal antifungal formulation in canine Malassezia dermatitis.

OBJECTIVE: Malassezia pachydermatis is a common cause of more widespread dermatitis in dogs (CMD). Recurrences are common, and this disorder can be very troubling for both dogs and for the pet owner. MATERIAL AND METHODS: The treatment of 20 dogs affected by dermatitis due to M. pachydermatis, with Malacalm(®), a commercially available mixture consisting of essential oils (Citrus aurantium 1%, Lavandula officinalis 1%, Origanum vulgare 0.5%, Origanum majorana 0.5%, Mentha piperita 0.5% and Helichrysum italicum var. italicum 0.5%, in sweet almond oil and coconut oil) is reported. The effectiveness of the whole mixture, of component essential oils and of their more represented compounds against clinical isolates was evaluated by a microdilution test. Twenty animals were topically administered the mixture twice daily for 1 month. Ten animals were treated with a conventional therapy based on ketoconazole 10mg/kg/day and chlorhexidine 2% twice a week for 3 weeks. At the end of both treatments animals significantly improved their clinical status. Adverse effects were never noticed. Follow-up visit performed on day 180th allowed to observe a recurrence of clinical signs in all the subjects treated conventionally, while not significant clinical changes were referred in dogs treated with Malacalm(®). The overall MIC value of Malacalm(®) was 0.3%. O. vulgare showed the lowest minimum inhibitory concentrations (MIC), being active at 0.8%, followed by M. piperita (1%), O. majorana (1.3%), C. aurantium (2%) and L. officinalis (4%) while H. italicum did not yield any antimycotic effect up to 10%. Active major compounds were thymol, carvacrol, p-cymene, 1,8-cineol, limonene and menthol. CONCLUSION: The phytotherapic treatment achieved a good clinical outcome, and no recurrence of skin disorders on day 180th was recorded. This herbal remedium appeared to be a safe tool for limiting recurrences of CMD.