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Intro: The ongoing outbreak of Monkeypoxvirus (MPXV) turned the spotlight back on zoonotic poxviruses (ZPs). MPXV, Cowpoxvirus (CPXV) and Vaccinia Virus (VV) are the zoonotic species belonging to the Orthopoxvirus (OPXV) genus in the Poxviridae family. The diagnostics currently available are designed for specialised laboratories and are unsuitable for field and point of care use. Here we present a sensitive and specific low-tech workflow for the rapid identification of ZPs. Methods: An isothermal (Hyperfluux) multiple dye assay, targeting the OPXV RNA Polimerase (RPO) was designed and validated. Analytical sensitivity was assessed by using both a sintetic dsDNA fragment and MPXV field samples. Standards were assigned to absolute values by using digital PCR and a probe- based assay. LOD was estimated by fitting a probit regression model with a prediction level of 95% after running a negative sample and 5 low positive samples in 5 replicates. Specificity was assessed by using bacterial and viral DNA from herpesviruses, zoonotic parapoxviruses and orthopoxviruses. For field applications, Xample Prep tool was employed to treat MPXV clinical samples prior amplification; the results were then compared to those obtained with DNAs extracted with standard method. Findings: The assay resulted highly sensitive with estimated LOD of 18,6 DNA copies/μl and rapid being performed in 35 minutes overall from samples to results. Only Zoonotic OPXV DNA was amplified showing a high specificity and comparable results were obtained from purified viral DNA and Xample Prep treated samples. Discussion: For its performances, rapidity, thermostable reagents, and ability to efficiently amplify clinical samples, without prior DNA extraction, the assay is suitable for use in low-resource settings, in the field and in community clinics. Conclusion: Besides clinical applications, to allow proper patients' management, the availability of such a diagnostic workflow can be critical to understand the real incidence and prevalence of zoonotic OPXV among humans and animal reservoirs.

Waves have the potential to increase the power output of a floating wind turbine by forcing its rotor to move against the wind. Starting from this observation, we use four multi-physics models of increasing complexity to investigate the role of waves and platform movements in the energy conversion process of four floating wind turbines of 5–15 MW in the Mediterranean Sea. Progressively adding realism to our simulations, we show that large along-wind rotor movements are needed to increase the power output of a floating wind turbine; however, these are prevented by the current technology of spar and semi-submersible platforms. Wind turbulence is the main cause of power fluctuations for the four floating wind turbines we examined and is preponderant over the effect of platform motions due to waves. In a realistic met-ocean environment, the power curve of the floating wind turbines we studied is lower than that obtained with a fixed foundation, with reductions in the annual energy production of 1.5 %–2.5 %. The lower energy production is mainly ascribed to the platform mean tilt, which reduces the rotor's effective area.

Objectives: In immunocompromised patients, asymptomatic Leishmania infection can reactivate, and evolve to severe disease. To date, no test is considered the gold standard for the identification of asymptomatic Leishmania infection. A combination of methods was employed to screen for Leishmania infection in patients undergoing kidney transplant (KT). Methods: We employed polymerase chain reaction for the detection of parasitic DNA in peripheral blood, Western blot to identify serum immunoglobulin G and whole blood assay to detect cytokines/chemokines after stimulation of whole blood with parasitic antigen. Results: One-hundred twenty patients residing in Italy were included in the study at the time of KT. Each patient that tested positive to at least one test was considered as Leishmania positive. Fifty out of 120 patients (42%) tested positive for one or more tests. The detection of specific cell-mediated response (32/111, 29%) was the most common marker of Leishmania infection, followed by a positive serology (24/120, 20%). Four patients (3%) harbored parasitic DNA in the blood. Conclusion: Our findings underline the high prevalence of asymptomatic Leishmania infection in patients undergoing KT in Italy, who are potentially at-risk for parasite reactivation and can benefit from an increased vigilance. Understanding the clinical relevance of these findings deserves further studies.

Exploiting by-products from the oenological industry to extract antioxidant chemicals is a shared goal that combines the need to reduce the wine sector’s environmental impact with the need to improve the availability of these biomolecules, according to a circular economy approach. Natural deep eutectic solvents (NaDES) have recently captured researchers’ interest as a safer and more environmentally friendly alternative to traditional solvents due to their effectiveness, low toxicity, and stability. In this work, we set out to investigate several NaDES for the extraction of phenolic chemicals from local monovarietal grape pomace resulting from different vinification procedures (including both red and rosé vinification of Negroamaro and Primitivo grapes; rosé vinification of Susumaniello grapes and white vinification of Chardonnay, Fiano and Malvasia bianca grapes), with the additional goal of generalizing the use of NaDES to extract chemicals of interest from organisms selected from the wide plant biodiversity. Three binary choline chloride-based NaDES (DES-Lac, DES-Tar, and DES-Gly, with lactic acid, tartaric acid, and glycerol as hydrogen bond donors, respectively) were compared to ethanol as a conventional solvent, and the extracts were evaluated using HPLC/MS and colorimetric techniques. The results revealed that each NaDES produces a substantially higher total phenolic yield than ethanol (up to 127.8 mg/g DW from Primitivo rosé grape pomace). DES-Lac and DES-Tar were more effective for anthocyanins extraction; the most abundant compound was malvidin 3-O-glucoside (highest extraction yield with DES-Lac from Susumaniello pomace: 29.4 mg/g DW). Regarding phenolic compounds, DES-Gly was the most effective NaDES producing results comparable to ethanol. Unexpectedly, Chardonnay pomace has the greatest content of astilbin. In most cases, grape pomace extracts obtained by rosé and white vinification provided the maximum yield. As a result, NaDES have emerged as a viable alternative to traditional organic solvent extraction techniques, allowing for higher (or equal) yields while significantly lowering costs, hazards, and environmental impact.

Staphylococcus argenteus, identified in 2006, represents a challenging case of bacterial taxonomic identification because of its high similarity to Staphylococcus aureus. In this context, neither mass spectrometry (MS) nor 16S gene analysis cannot precisely reveal the difference between the two species. In our study, the sensitivity to antibiotics of S. argenteus isolated from blood culture was tested, and the investigation of the bacterial genome was performed by Multi-Locus Sequence Typing (MLST) and Whole-Genome Next-Generation Sequencing (WG-NGS). The pathogen was identified as ST2250 and presented perfectly matched resistance genes, namely aph(3′)-III, mgrA, and sepA, whereas the virulence gene detected was scn. Two plasmids were found: the pSAS plasmid, belonging to the family of Inc18, and plasmid pN315, belonging to the Rep3 group. The epidemiological distribution and the spread of S. argenteus infection are scarcely documented, particularly when associated with sepsis. Therefore, a correct taxonomy identification, antibiogram, and resistance gene analysis may help in acquiring knowledge about this bacterium and implement its detection and treatment.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Background/Objectives. Adult medulloblastoma (AMB) patients should receive postoperative craniospinal irradiation (CSI) as a standard treatment. Volumetric intensity-modulated arc therapy (VMAT) is a promising method for CSI. This report summarizes the repositioning and dosimetric data outcomes for six AMB patients. Methods. Complete CSI and posterior cranial fossa irradiation, or tumor bed boost irradiation with Linac-based VMAT, was performed and evaluated. Patients were immobilized in the supine position with two thermoplastic masks (head-neck and abdomen). To ensure inter-fraction reproducibility during radiotherapy (RT), a single cone-beam CT (CBCT) scan for each isocenter and real-time surface-guided RT using AlignRT® were performed daily before and during the RT session. Match values of all three translational axes (x = lateral, y = longitudinal, z = vertical) were recorded. Results. From August 2022 to September 2023, six AMB patients were treated with CSI: three women and three men with a median age of 32 (22–42). All cases were classical MB, four were low risk, and two were defined as high risk due to the metastatic disease. All patients underwent surgery; two received a gross total resection. Low-risk patients received 36 Gy for CSI and a 54 Gy boost, while high-risk patients received 39 Gy for CSI. No significant toxicities greater than G2 were observed during RT, and only two cases reported decreased platelet counts. The dose to the organs at risk was low and acceptable. The mean dose to the heart, lungs, eyes, stomach, and thyroid were 4.4 Gy, 8.5 Gy, 12 Gy, 8.7 Gy, and 11 Gy, respectively. In terms of repositioning data, 124 CBCT scans were analyzed. Inter-fraction CBCT mean values for the study population in all translational directions were inferior to 2 mm in more than 90% of cases. Conclusions. VMAT is a convenient and effective treatment for AMB. Positioning and immobilization with masks (head and neck plus abdomen) reduce inter-fraction motion. [ABSTRACT FROM AUTHOR]

The impact of Xylella fastidiosa (Xf) subsp. pauca on the environment and economy of Southern Italy has been devastating. To restore the landscape and support the local economy, introducing new crops is crucial for restoring destroyed olive groves, and the almond tree (Prunus dulcis Mill. D. A. Webb) could be a promising candidate. This work focused on the resistance of the cultivar “Filippo Ceo” to Xf and evaluated its physiological and molecular responses to individual stresses (drought or pathogen stress) and combined stress factors under field conditions over three seasons. Filippo Ceo showed a low pathogen concentration (≈103 CFU mL−1) and a lack of almond leaf scorch symptoms. Physiologically, an excellent plant water status was observed (RWC 82–89%) regardless of the stress conditions, which was associated with an increased proline content compared to that of the control plants, particularly in response to Xf stress (≈8-fold). The plant’s response did not lead to a gene modulation that was specific to different stress factors but seemed more indistinct: upregulation of the LEA and DHN gene transcripts by Xf was observed, while the PR transcript was upregulated by drought stress. In addition, the genes encoding the transcription factors (TFs) were differentially induced by stress conditions. Filippo Ceo could be an excellent cultivar for coexistence with Xf subps. pauca, confirming its resistance to both water stress and the pathogen, although this similar health status was achieved differently due to transcriptional reprogramming that results in the modulation of genes directly or indirectly involved in defence strategies.

Orf virus (ORFV) belongs to the genus Parapoxvirus (Poxviridae family). It is the causative agent of contagious ecthyma (CE) that is an economically detrimental disease affecting small ruminants globally. Contagious ecthyma outbreaks are usually reported in intensive breeding of sheep and goats but they have also been reported in wildlife species. Notably, ORFV can infect humans, leading to a zoonotic disease. This study aims to elucidate the global evolutionary history of ORFV genomes in sheep and goats, including the first genomes from Central America in the analyses. In comparison to the last study on ORFV whole genomes, the database now includes 11 more sheep and goat genomes, representing an increase of 42%. The analysis of such a broader database made it possible to obtain a fine molecular dating of the coalescent time for ORFV S and G genomes, further highlighting the genetic structuring between sheep and goat genomes and corroborating their emergence in the latter half of 20th century.

Candida spp. are an important opportunistic pathogen that can represent a possible cause of severe infections, especially in immunocompromised individuals. The clinical impact of Candida spp. depends, in part, on the ability to form biofilms, communities of nestled cells into the extracellular matrix. In this study, we compared the biofilm formation ability of 83 strains of Candida spp. isolated from blood cultures and other materials, such as respiratory samples, urine, and exudate, and their sensitivity to fluconazole (FLZ). Strains were divided into tertiles to establish cut-offs to classify isolates as low, moderate, or high biofilm producers (0.839) and biofilms with low, moderate, or high metabolic activity (0.183). A non-linear relationship between biofilm production and metabolic activity was found in C. glabrata and C. tropicalis. In addition, the increase in minimum biofilm eradication concentrations (MBEC50) compared to the Minor Inhibitory Concentration (PMIC) of the planktonic form in Candida spp. confirms the role of biofilm in the induction of resistance to FLZ.

Grapevine “bois noir”, related to the presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), represents a serious threat in several vine-growing areas worldwide. In surveys conducted over two years, mild and/or moderate symptoms and lower pathogen titer were mainly associated with ‘Ca. P. solani’ strains harboring a secY gene sequence variant (secY52), whereas severe symptoms and higher titer were mainly observed in grapevines infected by phytoplasma strains carrying any one of another four variants. A comparison of amino acid sequences of the protein SecY of ‘Ca. P. solani’ strains revealed the presence of conservative and semi-conservative substitutions. The deduced three-dimensional (3D) protein analysis unveiled that one semi-conservative substitution identified in the sequence variant secY52 is responsible for a structural disordered region that probably confers a flexibility for binding to distinct molecular complexes. In fact, the other analyzed variants show an organized structure and the 3D in silico prediction allowed the identification of β-sheets. Thus, differences in symptom severity and pathogen concentration observed in grapevines infected by ‘Ca. P. solani’ strains carrying distinct secY gene sequence variants suggest a possible relationship between SecY protein structure and phytoplasma strain virulence.

The integration of sensing devices into cell culture systems is a topic of great interest in the study of pathologies and complex biological mechanisms in real-time. In particular, the fit-for-purpose microfluidic devices called organ-on-chip (OoC), which host living engineered organs that mimic in vivo conditions, benefit greatly from the integration of sensors, enabling the monitoring of specific chemical-physical parameters that can be correlated with biological processes. In this context, copper is an essential trace element whose total concentration may be associated with specific pathologies, and it is therefore important to develop reliable analytical techniques in cell systems. Copper can be determined by using the anodic stripping voltammetry (ASV) technique, but its applicability in cell culture media presents several challenges. Therefore, in this work, the performance of ASV in cell culture media was evaluated, and an acidification protocol was tested to improve the voltammetric signal intensity. A Transwell® culture model with Caco-2 cells was used to test the applicability of the developed acidification protocol by performing an off-line measurement. Finally, a microfluidic device was designed in order to perform the acidification of the cell culture medium in an automated manner and then integrated with a silicon microelectrode to perform in situ measurements. The resulting sensor-integrated microfluidic chip could be used to monitor the concentration of copper or other ions concentration in an organ-on-chip model; these functionalities represent a great opportunity for the non-destructive strategic experiments required on biological systems under conditions close to those in vivo.

“Bois noir” disease associated with ‘Candidatus Phytoplasma solani’ seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma–grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of ‘Ca. P. solani’-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in ‘Ca. P. solani-positive plants compared to -negative ones during the observed period.