Glutamine Supplementation as a Novel Metabolic Therapeutic Strategy for LIG3-Dependent Chronic Intestinal Pseudo-obstruction.

BACKGROUND & AIMS: We recently identified a recessive syndrome due to DNA ligase 3 (LIG3) mutations in patients with chronic intestinal pseudo-obstruction, leukoencephalopathy, and neurogenic bladder. LIG3 mutations affect mitochondrial DNA maintenance, leading to defective energy production. We aimed at identifying altered molecular pathways and developing possible targeted treatments to revert/ameliorate the cellular energy impairment. METHODS: Whole transcriptome analysis was performed on patient-derived fibroblasts total RNA and controls. Mitochondrial function, mitophagy, and l-glutamine supplementation effects were analyzed by live cell analysis, immunostaining, and Western blot. Patients were treated with Dipeptiven (Fresenius-Kabi) according to standard protocols. Patients' symptoms were analyzed by the Gastrointestinal Symptom Rating Scale questionnaire. RESULTS: We identified deregulated transcripts in mutant fibroblasts vs controls, including overexpression of genes involved in extracellular matrix development and remodeling and mitochondrial functions. Gut biopsy specimens of LIG3-mutant patients documented collagen and elastic fiber accumulation. Mutant fibroblasts exhibited impaired mitochondrial mitophagy indicative of dysfunctional turnover and altered Ca2+ homeostasis. Supplementation with l-glutamine (6 mmol/L), previously shown to increase mitochondrial DNA-defective cell survival, improved growth rate and adenosine 5'-triphosphate production in LIG3-mutant fibroblasts. These data led us to provide parenterally a dipeptide containing l-glutamine to 3 siblings carrying biallelic LIG3 mutations. Compared with baseline, gastrointestinal and extra-gastrointestinal symptoms significantly improved after 8 months of treatment. CONCLUSIONS: LIG3 deficiency leads to mitochondrial dysfunction. High levels l-glutamine supplementation were beneficial in LIG3-mutant cells and improved symptom severity without noticeable adverse effects. Our results provide a proof of concept to design ad hoc clinical trials with l-glutamine in LIG3-mutant patients.

Pro-aromatic Natural Terpenes as Unusual "Slingshot" Antioxidants with Promising Ferroptosis Inhibition Activity.

Ferroptosis is a cell death mechanism based on extensive cellular membrane peroxidation, implicated in neurodegenerative and other diseases. The essential oil component γ-terpinene, a natural monoterpene with a unique highly oxidizable pro-aromatic 1,4-cyclohexadiene skeleton, inhibits peroxidation of polyunsaturated lipid in model heterogeneous systems (micelles and liposomes). Upon H-atom abstraction, an unstable γ-terpinene-derived peroxyl radical is formed, that aromatizes to p-cymene generating HOO• radicals. As HOO• are small and hydrophilic radicals, they quickly diffuse outside the lipid core, blocking the radical chain propagation of polyunsaturated lipids. This unprecedented antioxidant "slingshot" mechanism explains why γ-terpinene shows a protective activity against ferroptosis, being effective at submicromolar concentrations in human neuroblastoma (SH-SY5Y) cells.

Myrcene: A Natural Compound Showing Anticancer Activity in HeLa Cells.

γ-terpinene, α-terpinene, p-cymene, and myrcene are monoterpenes found in many essential oils extracted from a variety of plants and spices. Myrcene also occurs naturally in plants such as hops, cannabis, lemongrass, and verbena and is used as a flavoring agent in food and beverage manufacturing. In this research, the biological efficacy of γ-terpinene, α-terpinene, p-cymene, and myrcene was studied in human cell lines (HeLa, SH-SY5Y, and HDFa). Cytotoxicity, cell proliferation, cell migration, and morphology assays were performed to obtain detailed information on the anticancer properties. Our results show that myrcene has potential biological activity, especially in HeLa cells. In this cell line, it leads to an arrest of proliferation, a decrease in motility and morphological changes with loss of sphericity and thickness, and DNA damage. In addition, the interaction of γ-terpinene, α-terpinene, p-terpinene, and myrcene with calf thymus DNA (ct-DNA) was studied by UV-visible spectrophotometry. DNA binding experiments show that only myrcene can interact with DNA with an apparent dissociation constant (Kd) of 29 × 10-6 M.

Energy and environmental assessment of plastic granule production from recycled greenhouse covering films in a circular economy perspective.

Plastic films can be considered as a high-value auxiliary material in agriculture with multiple important uses to fulfil, including covering films in greenhouse cultivation system. Such an application enables several benefits and, therefore, it is going through an important upsurge, especially in regions where protected crop cultivation is highly widespread. However, the increased demand for these covering films arouses concerns for their post-use treatment with regard to both the consumption of Non-Renewable Primary Energy (NRPE) resources and the emission of Greenhouse Gases (GHGs). Therefore, environmental analysis is needed to find and follow cleaner paths for the management and treatment of this kind of Agricultural Plastic Waste (APW), especially in the light of the gap currently existing in the specialised literature. In this context, this paper reports upon findings from a combined Life Cycle Assessment (LCA) of single environmental issues (i.e., energy and water consumption, and GHG emissions) applied to a Sicilian firm, representative of APW collection and recycling to obtain Low-Density Polyethylene (LDPE) granules. The results showed that electricity consumption for the whole recycling process is the most NRPE resource demanding and the most GHG emitting input item. Moreover, the washing phase of disused covering films is the highest water demanding within the recycling process. Potential improvements could be achieved by shifting from fossil energy source to renewable one. The installation of a wind power plant would lead to around 56% and 85% reduction in NRPE resource exploitation and GHG emission, respectively. Finally, despite the huge consumption of water and NRPE resources and the resulting GHG emissions, the production of recycled-LDPE granules is far more sustainable than the virgin counterpart.

Covering plastic films in greenhouses system: A GIS-based model to improve post use suistainable management.

Yearly, in Europe, more than 1 million tonnes of plastic materials are used in agricultural activities. Among the possible applications, plastic films for protected cultivation practices are highly used worldwide because of the significant advantage deriving from the shortening of the growing period. However, in the absence of a correct policy disposal of plastic films, environmental degradation could take place with serious ecological and economic consequences. In this study, a geographical information system (GIS) - based model to locate and quantify the yearly amount of agricultural plastic waste (APW) coming from crop-shelter coverage used in greenhouses system was put forward and was applied in a study area located in southern Italy, highly characterised by protected cultivation practices. Firstly, the areas with the highest density of crop shelters were mapped, then a suitable index to determine APW amount was computed and applied to obtain heat maps related to covering plastic films. Finally, sensitivity analyses were carried out by varying thickness, lifetime, and density of the covering films of the greenhouses, located in the considered samples. The index ranged between 976 kg ha-1yr-1and, 2484 kg ha-1yr-1. The results showed that the density of greenhouses and tunnels-greenhouses is still elevated nearby the coastline, highlighting that the guidelines of the territorial plan of the Province of Ragusa concerning the displacement of protected crops from the coast to the internal rural areas were disregarded. Moreover, the GIS-based model results could provide basic information for the analysis of the environmental impact due to transportation of APW. Therefore, these results could offer a suitable tool to improve the correct disposal management of covering plastic films and the related recycle policy.

Evaluation of biomethane potential from by-products and agricultural residues co-digestion in southern Italy.

The suitability of the co-digestion of feedstock-mixtures (by-products and agricultural residues) depends on their ability to produce biogas. In this study, the effects of mixing five feedstocks (citrus pulp, olive pomace, poultry manure, Italian sainfoin silage and opuntia fresh cladodes) on anaerobic digestion for biogas production have been investigated by carrying out biomethane potential (BMP) tests on six different mixing ratios of the selected five biomasses. The BMP test results demonstrated that all the six studied feedstock-mixtures could be potentially used for renewable energy generation by biogas plants. More in detail, two mixing ratios of the studied feedstock-mixtures showed the best biomethane potential of 249.9 and 260.1 Nm3CH4/tVS, respectively. Since this research study made it possible to screen the suitability and technical feasibility of the feedstock-mixtures analysed, the results provide the basis for subsequent pilot scale evaluation of anaerobic digestion in Mediterranean area, where by-products and agricultural residues are profuse and necessary to produce advanced biofuels.

GIS-based methodology for tracking the grazing cattle site use.

Interest in tracking and monitoring animals in livestock farming using wearable sensors has been steadily increasing. The use of these devices is particularly crucial in extensive livestock systems where direct interaction between animals and farmers is infrequent, necessitating strenuous efforts in long-distance herd monitoring. Internet of Things (IoT) technologies offer a promising solution to address the challenges posed by vast distances, enabling real-time and remote animal monitoring. In this study, an experimental trial was conducted using a custom-designed device, located in a Polyvinyl Chloride (PVC) case, specifically tailored to fit onto a collar. This case incorporates an integrated SigFox communication system, i.e., a Low Power Global Positioning System (LP-GPS) omnidirectional system, and a power supply. The trial took place in two grazing areas located in different territorial zones, designated as Case Study I and II. A LP-GPS collar was provided for each selected animal, and the data were recorded at 20-min intervals for Case Study I and 10-min intervals for Case Study II. The acquired data were then imported and analysed using Geographical Information Systems (GIS) software. Information was collected through a purpose-built web application (AppWeb). The objective was to analyze those territorial areas mostly occupied by animals within the two considered grazing areas by developing a GIS-based methodology. Specifically, customized algorithms such as Heatmap and Kernel Density Estimation (KDE) plugins were employed to conduct spatial analyses. The maps obtained through Heatmap plugin, showed the temporal-spatial distribution of animals within their grazing areas. Additionally, the KDE tool was used to classify preferred territorial areas, generating tailored charts for each animal in the sample. The individual Core Areas, determined through KDE evaluation for each animal, were overlaid to provide a comprehensive analysis of the monitored animals.The results achieved applying the GIS-based methodology facilitated the identification of animal positions and could be adopted to provide insights into feeding behavior and soil erosion, thereby aiding in the prevention of environmental issues.

Comprehensive review of technologies for separate digestate treatment and agricultural valorisation within circular and green economy.

Anaerobic digestion (AD) has the potential to catalyse the shift from a linear to a circular economy. However, effective treatment and management of both solid (DSF) and liquid (DLF) digestate fraction treatment and management require adopting sustainable technologies to recover valuable by-products like energy, biofuels, biochar, and nutrients. This study reviews state-of-the-art advanced technologies for DSF and DLF treatment and valorisation, using life cycle assessment (LCA) and techno-economic analysis (TEA) in integrated digestate management (IDM). Key findings highlight these technologies' potential in mitigating environmental impacts from digestate management, but there's a need to improve process efficiency, especially at larger scales. Future research should prioritize cost-effective and eco-friendly IDM technologies. This review emphasizes how LCA and TEA can guide decision-making and promote sustainable agricultural practices. Ultimately, sustainable IDM technologies can boost resource recovery and advance circular economy principles, enhancing the environmental and economic sustainability of AD processes.

LCA analysis for assessing environmenstal sustainability of new biobased chemicals by valorising citrus waste.

The global shift towards using biomass for biofuels and chemicals is accelerating due to increasing environmental concerns and geopolitical strategies. This study investigates a biorefinery model using citrus-processing-waste, specifically citrus pulp, to produce high-value products for various industries, including cosmetics, pharmaceuticals, flavours, fragrances, and food packaging. In Italy, particularly Sicily region, citrus processing generates significant amounts of waste, often improperly disposed of, contributing to environmental problems. Researchers have demonstrated that citrus waste can yield commercially valuable compounds. This study specifically focuses on orange peel waste (OPW), which constitutes about half of the fruit's weight, aiming to extract pectin and limonene through a combined process. The extraction process was carried out on a laboratory scale, and its sustainability was evaluated using a life cycle assessment (LCA) with SimaPro 8.1 software and the Impact 2002 + method. The functional unit adopted for this study is 300 g of OPW, obtained after the pre-treatment phase, from which 0.14 g of limonene and 8.22 g of pectin were extracted. The LCA results revealed that pectin extraction has a significantly higher environmental impact compared to limonene extraction, primarily due to the use of ethanol as a solvent, followed by electricity consumption. To mitigate this impact, the LCA assessed alternative, more sustainable solvents, resulting in a 73.4% reduction in the environmental footprint of the pectin extraction process. These findings underscore the critical role of LCA, even at the laboratory scale, in identifying environmental hotspots and providing insights for improving and optimizing processes for potential industrial-scale applications.

Combining Multiple Spectroscopic Techniques to Reveal the Effects of Staphylococcus aureus Infection on Human Bone Tissues.

Osteomyelitis (OM) and periprosthetic joint infections (PJIs) are major public health concerns in Western countries due to increased life expectancy. Infections usually occur due to bacterial spread through fractures, implants, or blood-borne transmission. The pathogens trigger an inflammatory response that hinders bone tissue regeneration. Treatment requires surgical intervention, which involves the precise removal of infected tissue, wound cleansing, and local and systemic antibiotic administration. Staphylococcus aureus (SA) is one of the most common pathogens causing infection-induced OM and PJIs. It forms antimicrobial-resistant biofilms and is frequently found in healthcare settings. In this proof-of-concept, we present an approach based on multiple spectroscopic techniques aimed at investigating the effects of SA infection on bone tissue, as well as identifying specific markers useful to detect early bacterial colonization on the tissue surface. A cross-section of a human femoral diaphysis, with negative-culture results, was divided into three parts, and the cortical and trabecular regions were separated from each other. Two portions of each bone tissue type were infected with SA for one and seven days, respectively. Multiple techniques were used to investigate the impact of the infection on bone tissue, Brillouin-Raman microspectroscopy and attenuated total reflection Fourier transform infrared spectroscopy were employed to assess and develop a new noninvasive diagnostic method to detect SA by targeting the bone of the host. The results indicate that exposure to SA infection significantly alters the bone structure, especially in the case of the trabecular type, even after just one day. Moreover, Raman spectral markers of the tissue damage were identified, indicating that this technique can detect the effect of the pathogens' presence in bone biopsies and pave the way for potential application during surgery, due to its nondestructive and contactless nature.

Enhancement of sustainable bioenergy production by valorising tomato residues: A GIS-based model.

In order to increase the utilization of renewable energy sources, the biomethane production through anaerobic digestion has notably developed over the last years. Although, it is worldwide recognized, that valorising waste resources (i.e., by-products) is an opportunity to improve the efficiency rate of the agro-industrial chains, by reducing economic and environmental impact, it is necessary to support the strategic planning development of a future sustainable biomethane chain in line to circular economy. In this study, by adopting network information technologies, the importance of a strategic planning for sustainable developing biomethane sector was highlighted, since feedstocks logistic and supply phase is a key-aspect of any bioenergy project. The developed Geographic Information Systems (GIS) - methodology, that could be applied in any territorial area considering any type of biomass, allowed to define suitable locations for allocating new anaerobic digestion plants with the aim of developing a sustainable valorisation of tomato residues, by minimising the economic and environmental impacts. The achieved results provided advanced knowledge for the literature on the topic, helping to develop an operational GIS-tool for defining sustainable strategies for planning new plants, and proved that the development of integrated approach to define sustainable strategies for resource management along the whole supply chain is crucial.

SARS-CoV-2 ORF3c impairs mitochondrial respiratory metabolism, oxidative stress, and autophagic flux.

Coronaviruses encode a variable number of accessory proteins that are involved in host-virus interaction, suppression of immune responses, or immune evasion. SARS-CoV-2 encodes at least twelve accessory proteins, whose roles during infection have been studied. Nevertheless, the role of the ORF3c accessory protein, an alternative open reading frame of ORF3a, has remained elusive. Herein, we show that the ORF3c protein has a mitochondrial localization and alters mitochondrial metabolism, inducing a shift from glucose to fatty acids oxidation and enhanced oxidative phosphorylation. These effects result in increased ROS production and block of the autophagic flux. In particular, ORF3c affects lysosomal acidification, blocking the normal autophagic degradation process and leading to autolysosome accumulation. We also observed different effect on autophagy for SARS-CoV-2 and batCoV RaTG13 ORF3c proteins; the 36R and 40K sites are necessary and sufficient to determine these effects.

[Assessing the impact of bladder ultrasound on catheter-associated urinary tract infections and health-care costs: an observational pre-post study]. / Valutazione dell'impatto dell'ecografia vescicale sulle infezioni del tratto urinario associate al catetere e sui costi sanitari: uno studio osservazionale pre-post.

. Assessing the impact of bladder ultrasound on catheter-associated urinary tract infections and health-care costs: an observational pre-post study. INTRODUCTION: The placement of a urinary catheter is a standard procedure, but it can cause discomfort, increase the risk of infections and costs. Pelvic ultrasound is a non-invasive assessment of the bladder and bladder catheter placement that can be performed by nurses. AIM: To assess the appropriateness of urinary catheter placement using bladder ultrasound, to monitor urinary catheter-associated urinary tract infections and costs associated with catheter use. METHODS: A single-centre pre-post observational study was conducted from September 2021 to August 2022 in patients aged >18 years requiring urinary monitoring; the first 6 months patients were assessed without the use of bladder ultrasound (control group), while the last 6 months with bladder ultrasound. RESULTS: 189 patients were included in the pre-ultrasound group and 175 patients in the post-ultrasound group; the demographic and clinical characteristics of the two groups were comparable. The rate of inappropriate catheterisation was 22.6% in the pre-group, whereas no inappropriate catheterisation was performed in the post-group. There was a 2.2% of absolute reduction in the rate of urinary tract infections (from 8.5% to 6.3%). Costs associated with the use of urinary catheters were reduced by 74.2% (from 173 to 44.8 euros). CONCLUSIONS: Bladder ultrasound in clinical practice is feasible and reduced the inappropriate use of bladder catheters, reducing patient risks and healthcare costs.

MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma.

BACKGROUND: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker. METHODS: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats. RESULTS: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells. CONCLUSIONS: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.

A Functional Minigenome of Parvovirus B19.

Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, characterized by a selective tropism for erythroid progenitor cells in bone marrow. Relevant information on viral characteristics and lifecycle can be obtained from experiments involving engineered genetic systems in appropriate in vitro cellular models. Previously, a B19V genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form, able to replicate and produce infectious viral particles in a producer/amplifier cell system. Based on such a system, we have now designed and produced a derived B19V minigenome, reduced to a replicon unit. The genome terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to include only the left-side genetic set, containing the coding sequence for the functional NS1 protein. Following transfection in UT7/EpoS1 cells, this minigenome still proved competent for replication, transcription and production of NS1 protein. Further, the B19V minigenome was able to complement B19-derived, NS1-defective genomes, restoring their ability to express viral capsid proteins. The B19V genome was thus engineered to yield a two-component system, with complementing functions, providing a valuable tool for studying viral expression and genetics, suitable to further engineering for purposes of translational research.

Assessing Influence Factors on Daily Ammonia and Greenhouse Gas Concentrations from an Open-Sided Cubicle Barn in Hot Mediterranean Climate.

Measurement of gas concentrations constitutes basic knowledge for the computation of emissions from livestock buildings. Although it is well known that hot climate conditions increase gas emissions, in the literature the relation between gas concentrations from open barns and animal-related parameters has not been investigated yet. This study aimed at filling this gap by evaluating daily gas concentrations within an open-sided barn in hot Mediterranean climate. The influence of microclimatic parameters (MC) and cow behavior and barn management (CBBM) were evaluated for ammonia (NH3), methane (CH4), and carbon dioxide (CO2) concentrations. Results showed that both MC and CBBM affected concentrations of NH3 (p < 0.02), CH4 (p < 0.001), and CO2 (p < 0.001). Higher values of NH3 concentration were detected during the cleaning of the floor by a tractor with scraper, whereas the lowest NH3 concentrations were recorded during animal lying behavior. Measured values of CO2 and CH4 were highly correlated (C = 0.87-0.89) due to the same sources of production (i.e., digestion and respiration). The different management of the cooling systems during the two observation periods reduced significantly CH4 concentrations in the barn when the cooling system in the feeding area was switched off. Based on methodological choices due to the specific barn typology, parameters related to animals can provide information on the variation of gas concentrations in the barn environment in hot climate conditions.

Co-digestion of by-products and agricultural residues: A bioeconomy perspective for a Mediterranean feedstock mixture.

This study focused on applying batch and continuous co-digestion approaches to investigate the effects of a feedstock mixture (FM) constituted by ten Mediterranean feedstocks highly available in the Mediterranean area (i.e., olive pomace, olive mill wastewater, citrus pulp, poultry litter, poultry and cattle manure, whey and cereal straw) on methane production for bioenergy generation. For the same feedstock mixture (FM), two different anaerobic digestion (AD) tests were carried out to evaluate the possible inhibitory effects of some biomasses on the biological process. The first AD test showed a methane yield equal to 229 Nm3CH4/tVS (27% lower than that measured during the batch test). During the second AD test, the specific production was 272 m3CH4/tVS. Both tests showed a similar methane content of methane in the biogas, equal to about 57%. The first AD test showed an inhibition effect of the process: total conversion of the organic matter into biogas was not ended. The second batch test demonstrated that the selected FM could be viable to carry out the co-digestion and could provide a flexible solution to generate advanced biofuels in biogas plants located in the Mediterranean area.

Anaerobic co-digestion of multiple agricultural residues to enhance biogas production in southern Italy.

To valorize agricultural wastes and byproducts in southern Italy, anaerobic co-digestion of six feedstocks (citrus pulp, olive pomace, cattle manure, poultry litter, whey, and corn silage) was studied to produce biogas for renewable energy generation. Both batch and semi-continuous co-digestion approaches were adopted to carry out the investigation. The feedstocks were mixed at different percentages according to their availabilities in southern Italy. The batch anaerobic co-digestion demonstrated that six studied feedstock mixtures generated an average of 239 mL CH4/g VS loading without significant difference between each other, which concluded that the feedstock mixtures can be used for biogas production. Considering the feedstock availability of citrus pulp and olive pomace in Sicily, three feedstock mixtures with the highest volatile solids concentration of citrus pulp (42% citrus pulp, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey; 34% citrus pulp, 8% olive pomace, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey; and 25% citrus pulp, 16% olive pomace, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey, respectively) were selected to run the semi-continuous anaerobic digestion. Under the stabilized culture condition, the feed mixture with 42% citrus pulp, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey presented the best biogas production (231 L methane/kg VS loading/day). The corresponding mass and energy balance concluded that all three tested feedstock mixtures have positive net energy outputs (1.5, 0.9, and 1.2 kWh-e/kg dry feedstock mixture, respectively).

Atypical squamous cells of undetermined significance-favour reactive compared to atypical squamous cells of undetermined significance-favour dysplasia: association with cervical intraepithelial lesions and human papillomavirus infection.

BACKGROUND AND OBJECTIVES: The current study compared the cervical cytological sub-category "atypical squamous cells of undetermined significance-favour reactive (AFR)", recently recommended to be eliminated by the Bethesda system, to the sub-category "atypical squamous cells of undetermined significance-favour dysplasia (ASC-US)", in terms of prevalence of coexistent squamous intraepithelial lesions of either low-grade (LSIL) or high-grade (HSIL) and rate of human papillomavirus (HPV) infection. STUDY DESIGN: One hundred women with AFR and 100 with ASC-US were consecutively included in the study. All patients underwent colposcopy, followed by biopsy when necessary, and were screened for HPV infection by the combined use of Hybrid Capture II (DIGENE) and PCR with MY09/11 primers, the latter followed by direct sequencing of the amplifications products for HPV genotyping. RESULTS: LSIL were detected in 5.6% of AFR and 18.5% of ASC-US (p=0.00812), HSIL only in 4.3% of ASC-US. HPV infection was diagnosed in 11.2% of AFR and 38.0% of ASC-US (p=0.00003); high-risk HPV types (namely, HPV-16, -18, -31, -66, -67 and -70) were found in 6.7% of AFR and 22.8% of ASC-US (p=0.00239). Evidence of HPV infection in absence of SIL was proven in 7.1% of AFR and in 22.5% of ASC-US (p=0.00622). CONCLUSION: The association of AFR with SIL and high-risk HPV infection is low but not inexistent. Thus, to avoid the risk of leaving some high-risk AFR patients untreated or without follow-up, it could be proposed to keep AFR as a cytological category and to triage it by HPV testing, similarly to what has been already recommended for ASC-US.