Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

BACKGROUND: Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. METHODS: One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. RESULTS: VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. CONCLUSIONS: This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

Vitamin Nutritional Status in Patients with Pancreatic Cancer: A Narrative Review.

Due to the high mortality rate in Western countries, pancreatic cancer is considered one of the big killers, leaving patients and their families with little hope upon diagnosis. Although surgical and drug therapies are critical for cancer patients to improve life expectancy and alleviation of suffering, nutrition plays a key role in improving cancer treatment outcomes. This narrative review, conducted as part of the activities of the Italian Society of Human Nutrition (SINU) working group in oncology, focuses on the prevalence of vitamin malnutrition among pancreatic cancer patients. The results of the literature search show that pancreatic cancer patients are at a heightened risk of water-soluble vitamin deficiencies, particularly of vitamins B1, B3, and B6. Additionally, they also face an increased risk of deficiency of fat-soluble vitamins. Among these vitamins, the potential role of vitamin D in pancreatic cancer has garnered the most attention, with its plasma levels being identified as a significant factor in patient survival. Investigating vitamin nutritional status could provide valuable insights for incorporating nutritional approaches into the prevention and treatment of pancreatic cancer, thereby reducing the exacerbation of symptoms associated with the diagnosis.

The importance of front-of-pack labels in improving health status and eating behavior.

The aim of this Editorial is to give an overview on the multiple aspects of front-of-pack labels (FOPLs) and provide the readers a balanced view of the problems raised in this research field, placing them in a wider context. Furthermore, this editorial paper discusses whether and how the use of FOPLs can promote health status in relation to the individual eating pattern and which should be the next research scenarios to investigate to further improve and integrate these tools.

Hairpin Structure Facilitates Multiplex High-Fidelity DNA Amplification in Real-Time Polymerase Chain Reaction.

Clinically and biologically, it is essential to detect rare DNA-sequence variants for early cancer diagnosis or drug-resistance mutation identification. Some of the common quantitative polymerase chain reaction (qPCR)-based variant detection methods are restricted in the limit of detection (LoD) because the DNA polymerases used for these methods have a high polymerase misincorporation rate; thus, the detection sensitivity is sometimes unsatisfactory. With the proofreading activity, high-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. However, there are currently no proper probe-based designs functioning as the fluorescence indicator allowing multiplexed HiFi qPCR reactions, thus restricting the application of HiFi DNA polymerases like the variant detection. We presented the occlusion system, composed of a 5'-overhanged primer with a fluorophore modification and a probe with a short-stem hairpin and a 3' quencher modification. We demonstrated that the occlusion system allowed multiplexing HiFi qPCR reaction, and it was compatible with the current variant-enrichment method to improve the LoD up to 10-fold. Thus, the occlusion system satisfactorily functioned as an efficient fluorescence indicator in HiFi qPCR reactions and allowed the application of HiFi DNA polymerases in variant detection methods to improve detection sensitivity.

The influence of front-of-pack nutritional labels on eating and purchasing behaviors: a narrative review of the literature.

BACKGROUND: Front-of-Pack Nutritional Labels are considered a useful tool to help consumers orient themselves in their food choices and direct their behavior toward a healthier diet. FOPNL development and use are part of a framework that includes cognitive, biological, hedonic and cultural aspects, able to affect consumers' eating and purchasing behavior. AIM: Given the complexity of the matter, the aim of this narrative review is to analyze the combination of different factors that drive food choices and eating behaviors and to highlight some aspects that are not fully studied. METHODS: The authors conducted the research using a top-down approach at first, followed by a bottom-up approach; starting with general considerations about the purchasing process, gradually narrowing the discussion to a specific sub-population, and finally extending the discussion back to more general reasonings about the direction to adopt in future, or at least to evaluate, for effective communication. RESULTS: Biases and attitudes toward food products were found to regularly interfere with buying behavior patterns, making it impossible to standardize an average consumer. This reflects in current research, increasing the complexity of the topic. All determinants influencing food choices are often assessed individually rather than in a synergistic and multidimensional context, while the purchasing scenario is characterized by multiple stimuli to which the consumer is subjected. FOPNLs' impact on perceived healthiness has been studied in different conditions, but some population subgroups have not been sufficiently represented. In particular, the effect of FOPNLs on consumers suffering from eating disorders is understudied and needs further attention. Furthermore, some approaches can be compared to "negative nutrition" or "loss-framed communication", putting nutrients out of context, emphasizing losses more than gains and risking promoting negative feelings in consumers. CONCLUSION: Due to the heterogeneity of studies, evidence on what works best in driving people to adopt lasting lifestyle changes is still mixed. Science communicators and policymakers should consider the possibility that a multi-component approach incorporating nutrition information and education may be a key strategy to promote consumers' self-consciousness and to support them in their cognitive efforts toward a healthy and sustainable diet. LEVEL OF EVIDENCE: Level V, narrative review.

Continuously tunable nucleic acid hybridization probes.

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Sensitive detection of cancer cells using light-mediated apta-PCR.

Apta-PCR is an ultrasensitive assay in which aptamers are exploited not only as biomolecular recognition elements, but also as reporter labels for amplification via real-time PCR. This methodology has been successfully applied to the detection of proteins, achieving limits of detection in the picomolar range. The introduction of caged aptamers that bear photo-labile groups, so called cages, at strategic positions so that their tertiary structure and thus their binding properties can be controlled by light, facilitates a more robust and attractive assay in terms of sample conservation and reusability. In this work, we report for the first time the use of caged aptamers for cell detection in an apta-PCR assay. Specifically, a sandwich format is used combining the capture of B-cells by an antibody with the specific detection of Burkitt's lymphoma cancer cells by a caged aptamer, acting as a reporter probe. Elution of the aptamer bound to the cancer cells is performed by light and the number of cells is then correlated with the amount of eluted caged aptamer using real-time PCR analysis. The reported technique shows an excellent sensitivity, achieving detection of as few as 77 cells, and due to the inherent robustness of the assay, this detection platform can be reused for further analyses, demonstrating potential applicability in proteomics and clinical diagnostics.

Validation of the Italian version of the Laval questionnaire: health-related quality of life in subjects with obesity.

BACKGROUND: Obesity is associated to increased risk of metabolic comorbidity as well as increased mortality. Notably, obesity is also associated to the impairment of the psychological status and of quality of life. Only three questionnaires are available in the Italian language evaluating the health-related quality of life in subjects with obesity. The aim of the present study was to test the validity and reliability of the Italian version of the Laval Questionnaire. METHODS: The original French version was translated into Italian and back-translated by a French native speaker. 273 subjects with obesity (Body Mass Index ≥ 30 kg/m2) were enrolled; the Italian version of the Laval Questionnaire and the O.R.Well-97 questionnaire were administered in order to assess health- related quality of life. The Laval questionnaire consists of 44 items distributed in 6 domains (symptoms, activity/mobility, personal hygiene/clothing, emotions, social interaction, sexual life). Disability and overall psychopathology levels were assessed through the TSD-OC test (SIO test for obesity correlated disabilities) and the SCL-90 (Symptom Checklist-90) questionnaire, respectively. To verify the validity of the Italian version, the analysis of internal consistency, test-retest reliability, and construct validity were performed. RESULTS: The observed proportion of agreement concordance of results was 50.2% with Cohen's K = 0.336 (CI 95%: 0.267-0.404), indicating a fair agreement between the two tests. Test-retest correlation was statistically significant (ρ = 0.82; p < 0.01); validity (standardized Chronbach's alpha) was considered reliable (α > 0.70). The analysis of construct validity showed a statistically significant association in terms of both total score (ρ = -0.66) and scores at each single domain (p < 0.01). A high correlation (p < 0.01) was observed between Laval questionnaire total and single domain scores and other related measures (Body Mass Index, TSD-OC scores, SCL-90 global severity index), revealing a high construct validity of the test. CONCLUSIONS: The Italian version of the Laval Questionnaire is a valid and reliable measure to assess the health-related quality of life in subjects with obesity.

Critical review of the equations predicting 6-minute walking distance in obese subjects.

The correlation between the 6-minute walking distance and obesity has been only partially explored. Results obtained from the existing predictive equations are influenced by a variety of factors (health status, severity of obesity, reduced muscle strength and mobility, decreased aerobic capacity, presence of comorbidities). The aim of our study was to verify, compare and discuss the 6MWD predictive capacity of the equations currently available in the literature in a sample of obese subjects. Despite similar study design, the considered articles varied with regard to the number of individuals included and to age range. None of the equations available in the literature was able to accurately predict the six-minute walked distance performed by the obese subjects involved in our study. The six-minute walked distance performed by obese subjects seems to be influenced by other factors than the variables included in the equations from the literature, suggesting that they may not thoroughly capture the complexity of disability in obese people. Future research should be designed in order to validate population-specific equations, by including variables related to clinical, functional, and psychological areas, which are usually impaired in obese individuals.

Label-free detection of gliadin food allergen mediated by real-time apta-PCR.

Celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten. The only effective treatment consists in a lifelong gluten-free diet, requiring the food industry to tightly control the gluten contents of their products. To date, several gluten quantification approaches using antibodies are available and recommended by the legal authorities, such as Codex Alimentarius. However, whilst these antibody-based tests exhibit high sensitivity and specificity, the production of antibodies inherently requires the killing of host animals and is time-consuming and relatively expensive. Aptamers are structured single-stranded nucleic acid ligands that bind with high affinity and specificity to their cognate target, and aiming for a cost-effective viable alternative to the use of antibodies. Herein, we report the systematic evolution of ligands by exponential enrichment (SELEX)-based selection of a DNA aptamer against gliadin from a combinatorial DNA library and its application in a novel detection assay. Taking into account the hydrophobic nature of the gliadin target, a microtitre plate format was exploited for SELEX, where the target was immobilised via hydrophobic interactions, thus exposing aptatopes accessible for interaction with the DNA library. Evolution was followed using surface plasmon resonance, and following eight rounds of SELEX, the enriched DNA pool was cloned, sequenced and a clear consensus motif was identified. An apta-PCR assay was developed where competition for the aptamer takes place between the surface-immobilised gliadin and gliadin in the target sample, akin to an ELISA competitive format where the more target present in the sample, the less aptamer will bind to the immobilised gliadin. Following competition, any aptamer bound to the immobilised gliadin was heat-eluted and quantitatively amplified using real-time PCR, achieving a detection limit of approx. 2 nM (100 ng mL(-1)). The specificity of the selected aptamer was demonstrated and no cross-reactivity was observed with streptavidin, bovine serum albumin or anti-gliadin IgG.

Environmental and economic sustainability of the Mediterranean Diet.

The Mediterranean diet (MD) has been proposed as a healthy dietary pattern for disease prevention. However, little information exists on the cost and on the environmental impact of such a dietary model. We compared the environmental impact and the costs of the current food consumption pattern of the Italian population and the Mediterranean model in order to investigate its overall sustainability. The environmental impact was calculated on the basis of three indexes, i.e. Carbon, Ecological and Water Footprint. The costs (Euro) per person of the MD and of the current Italian household food expenditure were considered on a weekly basis according to the 2013 data from the Observatory prices and tariffs of the Ministry of Economic Development and the service SMS consumers of the Ministry of Agriculture, Food and Forestry. The MD resulted to produce a lower environmental impact than the current food consumption of the Italian population. The monthly expenditure of the MD is slightly higher in the overall budget compared to the current expenditure allocated to food by the Italian population, but there is a substantial difference in the distribution of budget according to the different food groups.

Obesity treatment: results after 4 years of a Nutritional and Psycho-Physical Rehabilitation Program in an outpatient setting.

INTRODUCTION: Obesity is a chronic disease as well as a risk factor for cardiovascular, metabolic and osteoarticular diseases, affecting the psychosocial health and the quality of life. Recent evidence suggests that the adequate treatment of obesity should provide a multidimensional multidisciplinary approach including nutritional therapy, psycho-educational classes and physical reconditioning/motor rehabilitation. The aim of this approach should be to maintain the results over time with a positive impact on the obesity-related cardiovascular and metabolic risk. AIM OF THE STUDY: To evaluate the effectiveness of a multidisciplinary Nutritional and Psycho-Physical Reconditioning Program (NPPRP) in an outpatient setting. MATERIALS AND METHODS: The observational prospective cohort study involved subjects, aged from 18 to 65 years, with a BMI >30 kg/m(2), who were followed up over 48 months. After the first nutritional and psychological examination, patients who refused NPPRP were treated according to standard nutrition procedures (SNT). Patients were followed through monthly medical examinations and then by annual telephonic structured interviews for 4 years. Changes in body weight, eating behavior, physical activity performance, and the occurrence of clinical obesity-related complications were considered as outcome measures. RESULTS: Of 464 enrolled patients, 161 (34.7%) took part in the follow-up. From the enrollment to the end of follow-up, weight loss was greater in the NPPRP group than in the SNP group (-8.08 ± 10 kg versus -3.0 ± 6 kg). After 4 years eating behavior improved in both groups. The percentage of patients who continued to perform physical activity was higher in the NPPRP group than in the SNT group (61.0 versus 34.1%). The SNT group reported complications more frequently than the NPPRP group: hypertension (19 versus 5.8%), dyslipidemia (19.4 versus 12.7%), and skeletal problems (26.9 versus 17.5%). The main reasons for drop-out from the rehabilitation program were logistic problems (distance or time) in both groups; disappointment was higher in the SNT group than in the NPPRP group (37.8 versus 15.6%). CONCLUSION: A multidimensional multidisciplinary approach including nutritional intervention and psycho-physical rehabilitation, set against a conventional diet therapy, was more effective in the long-term outcome of obesity with regard to weight loss, physical activity, possible eating disorders, and obesity-related complications.

Conventional Radiology Evaluation of Neonatal Intravascular Devices (NIVDs): A Case Series.

Our radiology department conducted an assessment of 300 neonatal radiographs in the neonatal intensive care unit over almost two years. The purpose was to evaluate the correct positioning of intravascular venous catheters. Our case series revealed that out of a total of 95 cases with misplaced devices, 59 were umbilical venous catheters and 36 were peripherally inserted central catheters. However, all of the central venous catheters were found to be properly positioned. Misplacements of neonatal intravascular devices were found to occur more frequently than expected. The scientific literature contains several articles highlighting the potential complications associated with misplaced devices. Our goal is to highlight the potential misplacements and associated complications that radiologists may encounter while reviewing conventional radiology imaging. Based on our experience, which primarily involved placing UVCs and PICCs, we discovered that conventional radiology is the most effective method for assessing proper device placement with the lowest possible radiation exposure. Given the high number of neonatal vascular device placement procedures, it is essential for radiologists to maintain a high level of vigilance and stay updated on the latest developments in this field.

Food Compass and the challenge of sustainability on the route towards healthful diets.

In order to tackle the global increase in overweight and obesity prevalence, several nutrient profiling systems have been developed; among others, Food Compass Score (FCS) has been designed to encompass multiple domains of food healthfulness. However, environmental sustainability of healthy diets is another crucial dimension which should not be overlooked in the context of human health. The aim of the present study is to assess the association between healthiness and environmental sustainability of food items, using the FCS and Agribalyse databases, respectively. A total of 806 matching food items were identified, grouped in 12 food categories; within each category, differences in median Z-scores between FCS and Single Environmental Footprint (EF) Score were assessed. While Fruits, Legumes and Nuts, Mixed foods, Meat Poultry and Eggs (MPE), Savory and Sweets, and Vegetables showed statistically significant differences (all p < 0.001), Beverages (p = 0.361), Dairy (p = 0.092), Fats and Oils (p = 0.594), Grains (p = 0.436), Sauce and Condiments (p = 0.093), and Seafood (p = 0.241) had similar Food Compass and Single EF Z-scores distributions. These findings underscore a relevant lack of difference between healthfulness and environmental impact of some prominent food categories, such as Grains and Seafood. Therefore, we suggest matching nutrient profiling systems with adequate environmental sustainability indices.

All for one: Collaboration between dermatologist, radiation oncologist and radiologist in the clinical management of "difficult to treat" non melanoma skin cancer.

This series introduces the clinical management of difficult-to-treat non-melanoma skin cancers (NMSCs) through a multidisciplinary approach, emphasizing the integration of dermoscopy and Ultra high-frequency ultrasound (UHFUS) for accurate diagnosis and treatment planning, particularly in cases referred for radiotherapy (RT). Dermoscopy aids in diagnosing both pigmented and non-pigmented skin lesions, guiding treatment margins and reducing the benign-to-malignant biopsy ratio. UHFUS provides valuable insights into tumor size, depth, and vascularity, complementing clinical evaluations and assisting in RT planning. Three challenging cases are presented, highlighting the pivotal role of dermoscopy and UHFUS in decision-making and treatment optimization. Collaboration between dermatologists, radiation oncologists, and radiologists enhances diagnostic accuracy, tailoring treatment plans to individual patient needs and preferences, ultimately improving patient outcomes and experience. The integration of these imaging techniques holds promise for optimizing non-surgical treatments like RT and monitoring treatment progress, offering a personalized approach to NMSC management.

Assessing the future global distribution of land ecosystems as determined by climate change and cropland incursion.

The geographic distribution of natural ecosystems is affected by both climate and cropland. Discussions of future land use/land cover usually focus on how cropland expands and displaces natural vegetation especially as climate change impacts become stronger. Less commonly considered is the direct influence of climate change on natural ecosystems simultaneously with cropland incursion. We combine a natural vegetation model responsive to climate with a cropland allocation algorithm to assess the relative importance of climate change compared to cropland incursion. Globally, the model indicates that climate change drives larger gains and losses than cropland incursion. For example, in the Amazonian rainforests, more than one sixth of the forest area could be lost due to climate change with cropland playing virtually no role. Our findings suggest that policies to protect specific ecosystems may be undercut by climate change and that localized analyses that fully account for the impacts of a changing climate on natural vegetation and agriculture are necessary to formulate policies that preserve natural ecosystems over the long term. Supplementary Information: The online version contains supplementary material available at 10.1007/s10584-023-03584-3.

The role of neighborhood inequalities on diabetes prevention care: a mini-review.

An emerging research niche has focused on the link between social determinants of health and diabetes mellitus, one of the most prevalent non-communicable diseases in modern society. The aim of the present mini-review is to explore and summarize current findings in this field targeting high-income countries. In the presence of disadvantaged neighborhood factors (including socioeconomic status, food environment, walkability and neighborhood aesthetics), diabetes prevention and care are affected at a multidimensional level. The vast majority of the included studies suggest that, besides individual risk factors, aggregated neighborhood inequalities should be tackled to implement effective evidence-based policies for diabetes mellitus.

Preaxial polydactyly of the foot in an adult patient diagnosed by X-ray after a trauma.

Polydactyly, or hyperdactyly, is a frequent malformation, with a reported incidence between 0.37 and 1.2 per 1000 live births. Most cases encountered in medical practice are sporadic cases, usually presenting one-sided manifestations. More frequently, polydactyly can be detected prenatally through an ultrasound examination, if not, it is usually diagnosed at birth, during the first physical examination. Although the clinical diagnosis is relatively easy in patients with manifest cases, it can sometimes be present with mild or partial forms that are not clinically evident on physical examination, resulting in later diagnosis and treatment. We reported a particular case of polydactyly of the foot not clinically manifest, diagnosed in a 39-year-old Caucasian Male patient with a history of recurrent localized pain in the big toe often associated with subungual bleeding, since he was a child who came to our emergency room following a car accident. Polydactyly is a frequently reported congenital malformation which may present in many different varieties of deformities. In this case, the X-ray, which was required after a car accident, leads to the incidental diagnosis of polydactyly in an adult patient. As described, because of the functional limitations related to this malformation, as well as to limit recurrent pain, and subungual anomalies, the patient underwent to a surgical correction to improve its quality of life.