SARS-CoV-2 RNA viability on high-touch surfaces and evaluation of a continuous-flow ozonation treatment.

Background: The COVID-19 emergency has highlighted the importance of prevention systems and environ-mental microbiological monitoring as fundamental elements in the response to epidemics and other such threats to individual and collective health. The use of automated "No-touch" room disinfection systems eliminates or reduces the dependence on operators, thus allowing an improvement in the effectiveness of terminal disinfection. Study design: In the present study, we focused on possible SARS-CoV-2 contamination of surfaces of com-mercial services, and the effectiveness of ozone treatment on the virus. Methods: Analyses were conducted on 4-7 October and 27-30 December 2021 in four supermarkets in an Apulian city; supermarkets A and B were equipped with an ozonisation system, while C and D were without any environmental remediation. Results: SARS-CoV-2 RNA was detected by real-time RT-PCR only in December, in 6% of the surfaces tested, and all examined samples were found to be negative after viral culture, since no cytopathic effect was observed. A statistically significant difference emerged from the comparison of October vs. December (p = 0.0289), but no statistically significant difference (p = 0.6777) emerged from the comparison between supermarkets with and without the ozonisation system. Conclusions: Although no important changes were observed by treating the environments with ozonisation systems, further studies are needed to validate the effectiveness of environmental treatments with airborne disinfectants.

Rapid screening for SARS-CoV-2 variants of concern in clinical and environmental samples using nested RT-PCR assays targeting key mutations of the spike protein.

New SARS-CoV-2 mutations are constantly emerging, raising concerns of increased transmissibility, virulence or escape from host immune response. We describe a nested RT-PCR assay (~1500 bps) to detect multiple nucleotide changes resulting in key spike protein mutations distinctive of the major known circulating SARS-CoV-2 variants, including the three Variants of Concern (VOCs) 20I/501Y.V1 (United Kingdom), 20H/501Y.V2 (South Africa), and 20 J/501Y.V3 (Brazil), as well as the 20E.EU1 variant (Spain), the CAL.20C recently identified in California, and the mink-associated variant (GR, lineage B.1.1.298). Prior to application to field samples, the discriminatory potential of this PCR assay was explored using GISAID and Nextclade. To extend variant detection to challenging matrices such as sewage, where the amplification of long fragments is problematic, two short nested RT-PCR assays (~300 bps) were also designed, targeting portions of the region spanned by the long nested assay. The three newly-designed assays were then tested on field samples, including 31 clinical samples (7 fully-sequenced swab samples, and 24 uncharacterized ones) and 34 urban wastewater samples, some of which collected in areas where circulation of VOCs had been reported. The long assay successfully amplified 29 of the 31 swabs (93%), allowing the correct identification of variants 20I/501Y.V1 and 20E.EU1 present in the panel of previously characterized samples. The Spanish variant was detected in 14/24 of the uncharacterized samples as well. The sequences obtained using the short assays were consistent with those obtained with the long assay. Mutations characteristic of VOCs (UK and Brazilian variant) and of other variant (Spanish) were detected in sewage samples. To our knowledge, this is the first evidence of the presence of sequences harboring key mutations of 20I/501Y.V1 and 20 J/501Y.V3 in urban wastewaters, highlighting the potential contribution of wastewater surveillance to explore SARS-CoV-2 diversity. The developed nested RT-PCR assays can be used as an initial rapid screening test to select clinical samples containing mutations of interest. This can speed up diagnosis and optimize resources since it allows full genome sequencing to be done only on clinically relevant specimens. The assays can be also employed for a rapid and cost-effective detection of VOCs or other variants in sewage for the purposes of wastewater-based epidemiology. The approach proposed here can be used to better understand SARS-CoV-2 variant diversity, geographic distribution and impact worldwide.

Nanoscale 3D quantitative imaging of 1.88 Ga Gunflint microfossils reveals novel insights into taphonomic and biogenic characters.

Precambrian cellular remains frequently have simple morphologies, micrometric dimensions and are poorly preserved, imposing severe analytical and interpretational challenges, especially for irrefutable attestations of biogenicity. The 1.88 Ga Gunflint biota is a Precambrian microfossil assemblage with different types and qualities of preservation across its numerous geological localities and provides important insights into the Proterozoic biosphere and taphonomic processes. Here we use synchrotron-based ptychographic X-ray computed tomography to investigate well-preserved carbonaceous microfossils from the Schreiber Beach locality as well as poorly-preserved, iron-replaced fossil filaments from the Mink Mountain locality, Gunflint Formation. 3D nanoscale imaging with contrast based on electron density allowed us to assess the morphology and carbonaceous composition of different specimens and identify the minerals associated with their preservation based on retrieved mass densities. In the Mink Mountain filaments, the identification of mature kerogen and maghemite rather than the ubiquitously described hematite indicates an influence from biogenic organics on the local maturation of iron oxides through diagenesis. This non-destructive 3D approach to microfossil composition at the nanoscale within their geological context represents a powerful approach to assess the taphonomy and biogenicity of challenging or poorly preserved traces of early microbial life, and may be applied effectively to extraterrestrial samples returned from upcoming space missions.

Effect of ferric citrate on amyloid-beta peptides behavior.

Amyloid beta (Aß) aggregation and oxidative stress are two of the central events in Alzheimer's Disease (AD). Both these phenomena can be caused by the interaction of Aß with metal ions. In the last years the interaction between ZnII , CuII , and Aß was much studied, but between iron and Aß it is still little known. In this work we determine how three Aß peptides, present in AD, interact with FeIII -citrate. The three Aß peptides are: full length Aß1-42, an isoform truncated at Glutamic acid in position three, Aß3-42, and its pyroglutamated form AßpE3-42. Conformation and morphology of the three peptides, aggregated with and without FeIII -citrate were studied. Besides, we have determined the strength of the interactions Aß/FeIII -citrate studying the effect of ethylenediaminetetraacetic acid as chelator. Results reported here demonstrate that FeIII -citrate promotes the aggregation in all the three peptides. Moreover, Aspartic acid 1, Glutamic acid 3, and Tyrosine 10 have an important role in the coordination with iron, generating a more stable complex for Aß1-42 compared to that for the truncated peptides.

First report of Coxiella burnetii and Borrelia burgdorferi sensu lato in poultry red mites, Dermanyssus gallinae (Mesostigmata, Acari), related to urban outbreaks of dermatitis in Italy.

The poultry red mite (PRM), Dermanyssus gallinae, is a nonburrowing haematophagous nest-dwelling ectoparasite of birds; occasionally it bites humans, inducing dermatitis. The possibility that this parasite may also be involved in transmission of pathogens is an additional concern. We investigated the presence of zoonotic agents in PRMs from bird nests and pets, and related them to urban outbreaks of dermatitis. A total of 98 PRMs from 12 outbreaks of PRM dermatitis that occurred in Italian cities from 2001 to 2017 were molecularly investigated for detection of Coxiella spp. (16S rRNA), Chlamydophila spp. (16S rRNA), Rickettsia spp. (17 kDa protein-encoding gene), Borrelia burgdorferi sensu lato (groEL gene) and Bartonella spp. (16S-23S rRNA intergenic spacer). Of the 12 tested mite pools, one was positive for Coxiella burnetii (100% identity) and two for B. burgdorferi sensu lato (99% with Borrelia afzelii). For the first time, the presence of B. burgdorferi sensu lato and C. burnetii is reported in PRMs from urban areas. Birds, mainly pigeons, can harbour both pathogens. Therefore, birds and their nest-dwelling PRMs may play a role in the epidemiology of these infections.

Geometry-dependent DNA-TiO2 immobilization mechanism: A spectroscopic approach.

DNA nucleotides are used as a molecular recognition system on electrodes modified to be applied in the detection of various diseases, but immobilization mechanisms, as well as, charge transfers are not satisfactorily described in the literature. An electrochemical and spectroscopic study was carried out to characterize the molecular groups involved in the direct immobilization of DNA structures on the surface of nanostructured TiO2 with the aim of evaluating the influence of the geometrical aspects. X-ray photoelectron spectroscopy at O1s and P2p core levels indicate that immobilization of DNA samples occurs through covalent (POTi) bonds. X-ray absorption spectra at the Ti2p edge reinforce this conclusion. A new species at 138.5eV was reported from P2p XPS spectra analysis which plays an important role in DNA-TiO2 immobilization. The POTi/OTi ratio showed that quantitatively the DNA immobilization mechanism is dependent on their geometry, becoming more efficient for plasmid ds-DNA structures than for PCR ds-DNA structures. The analysis of photoabsorption spectra at C1s edge revealed that the molecular groups that participate in the C1s→LUMO electronic transitions have different pathways in the charge transfer processes at the DNA-TiO2 interface. Our results may contribute to additional studies of immobilization mechanisms understanding the influence of the geometry of different DNA molecules on nanostructured semiconductor and possible impact to the charge transfer processes with application in biosensors or aptamers.

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum.

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.

The effect of age and sex on facial mimicry: a three-dimensional study in healthy adults.

To assess sex- and age-related characteristics in standardized facial movements, 40 healthy adults (20 men, 20 women; aged 20-50 years) performed seven standardized facial movements (maximum smile; free smile; "surprise" with closed mouth; "surprise" with open mouth; eye closure; right- and left-side eye closures). The three-dimensional coordinates of 21 soft tissue facial landmarks were recorded by a motion analyser, their movements computed, and asymmetry indices calculated. Within each movement, total facial mobility was independent from sex and age (analysis of variance, p>0.05). Asymmetry indices of the eyes and mouth were similar in both sexes (p>0.05). Age significantly influenced eye and mouth asymmetries of the right-side eye closure, and eye asymmetry of the surprise movement. On average, the asymmetry indices of the symmetric movements were always lower than 8%, and most did not deviate from the expected value of 0 (Student's t). Larger asymmetries were found for the asymmetric eye closures (eyes, up to 50%, p<0.05; mouth, up to 30%, p<0.05 only in the 20-30-year-old subjects). In conclusion, sex and age had a limited influence on total facial motion and asymmetry in normal adult men and women.