Emerging Microfluidic Tools for Simultaneous Exosomes and Cargo Biosensing in Liquid Biopsy: New Integrated Miniaturized FFF-Assisted Approach for Colon Cancer Diagnosis.

The early-stage diagnosis of cancer is a crucial clinical need. The inadequacies of surgery tissue biopsy have prompted a transition to a less invasive profiling of molecular biomarkers from biofluids, known as liquid biopsy. Exosomes are phospholipid bilayer vesicles present in many biofluids with a biologically active cargo, being responsible for cell-to-cell communication in biological systems. An increase in their excretion and changes in their cargo are potential diagnostic biomarkers for an array of diseases, including cancer, and they constitute a promising analyte for liquid biopsy. The number of exosomes released, the morphological properties, the membrane composition, and their content are highly related to the physiological and pathological states. The main analytical challenge to establishing liquid biopsy in clinical practice is the development of biosensors able to detect intact exosomes concentration and simultaneously analyze specific membrane biomarkers and those contained in their cargo. Before analysis, exosomes also need to be isolated from biological fluids. Microfluidic systems can address several issues present in conventional methods (i.e., ultracentrifugation, size-exclusion chromatography, ultrafiltration, and immunoaffinity capture), which are time-consuming and require a relatively high amount of sample; in addition, they can be easily integrated with biosensing systems. A critical review of emerging microfluidic-based devices for integrated biosensing approaches and following the major analytical need for accurate diagnostics is presented here. The design of a new miniaturized biosensing system is also reported. A device based on hollow-fiber flow field-flow fractionation followed by luminescence-based immunoassay is applied to isolate intact exosomes and characterize their cargo as a proof of concept for colon cancer diagnosis.

Field-Flow Fractionation in Molecular Biology and Biotechnology.

Field-flow fractionation (FFF) is a family of single-phase separative techniques exploited to gently separate and characterize nano- and microsystems in suspension. These techniques cover an extremely wide dynamic range and are able to separate analytes in an interval between a few nm to 100 µm size-wise (over 15 orders of magnitude mass-wise). They are flexible in terms of mobile phase and can separate the analytes in native conditions, preserving their original structures/properties as much as possible. Molecular biology is the branch of biology that studies the molecular basis of biological activity, while biotechnology deals with the technological applications of biology. The areas where biotechnologies are required include industrial, agri-food, environmental, and pharmaceutical. Many species of biological interest belong to the operational range of FFF techniques, and their application to the analysis of such samples has steadily grown in the last 30 years. This work aims to summarize the main features, milestones, and results provided by the application of FFF in the field of molecular biology and biotechnology, with a focus on the years from 2000 to 2022. After a theoretical background overview of FFF and its methodologies, the results are reported based on the nature of the samples analyzed.

FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies.

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices.

The Challenges of O2 Detection in Biological Fluids: Classical Methods and Translation to Clinical Applications.

Dissolved oxygen (DO) is deeply involved in preserving the life of cellular tissues and human beings due to its key role in cellular metabolism: its alterations may reflect important pathophysiological conditions. DO levels are measured to identify pathological conditions, explain pathophysiological mechanisms, and monitor the efficacy of therapeutic approaches. This is particularly relevant when the measurements are performed in vivo but also in contexts where a variety of biological and synthetic media are used, such as ex vivo organ perfusion. A reliable measurement of medium oxygenation ensures a high-quality process. It is crucial to provide a high-accuracy, real-time method for DO quantification, which could be robust towards different medium compositions and temperatures. In fact, biological fluids and synthetic clinical fluids represent a challenging environment where DO interacts with various compounds and can change continuously and dynamically, and further precaution is needed to obtain reliable results. This study aims to present and discuss the main oxygen detection and quantification methods, focusing on the technical needs for their translation to clinical practice. Firstly, we resumed all the main methodologies and advancements concerning dissolved oxygen determination. After identifying the main groups of all the available techniques for DO sensing based on their mechanisms and applicability, we focused on transferring the most promising approaches to a clinical in vivo/ex vivo setting.

Application of Af4-Multidetection to Liraglutide in Its Formulation: Preserving and Representing Native Aggregation.

Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, particularly when the API is marketed as a ready-to-use solution. Consequently, appropriate non-destructive techniques able to replicate the peptide environment must be employed. In our work, we exploited Asymmetrical Flow Field-Flow Fractionation (AF4), connected to UV, dRI, fluorescence, and MALS detectors, to fully characterize the aggregation state of Liraglutide, a peptide API used for the treatment of diabetes type 2 and chronic obesity. In previous studies, Liraglutide was hypothesized to assemble into hexa-octamers in phosphate buffer, but no information on its behavior in the formulation medium was provided up to now. The method used allowed researchers to work using formulation as the mobile phase with excellent recoveries and LoQ/LoD, discerning between stable and degraded samples, and detecting, when present, aggregates up to 108 Da. The native state of Liraglutide was assessed and found to be an association into pentamers, with a non-spherical conformation. Combined to benchmark analyses, the sameness study was complete and descriptive, also giving insight on the aggregation process and covalent/non-covalent aggregate types.

A Green Analytical Method Combined with Chemometrics for Traceability of Tomato Sauce Based on Colloidal and Volatile Fingerprinting.

Tomato sauce is a world famous food product. Despite standards regulating the production of tomato derivatives, the market suffers frpm fraud such as product adulteration, origin mislabelling and counterfeiting. Methods suitable to discriminate the geographical origin of food samples and identify counterfeits are required. Chemometric approaches offer valuable information: data on tomato sauce is usually obtained through chromatography (HPLC and GC) coupled to mass spectrometry, which requires chemical pretreatment and the use of organic solvents. In this paper, a faster, cheaper, and greener analytical procedure has been developed for the analysis of volatile organic compounds (VOCs) and the colloidal fraction via multivariate statistical analysis. Tomato sauce VOCs were analysed by GC coupled to flame ionisation (GC-FID) and to ion mobility spectrometry (GC-IMS). Instead of using HPLC, the colloidal fraction was analysed by asymmetric flow field-fractionation (AF4), which was applied to this kind of sample for the first time. The GC and AF4 data showed promising perspectives in food-quality control: the AF4 method yielded comparable or better results than GC-IMS and offered complementary information. The ability to work in saline conditions with easy pretreatment and no chemical waste is a significant advantage compared to environmentally heavy techniques. The method presented here should therefore be taken into consideration when designing chemometric approaches which encompass a large number of samples.

Perceived work ability at return to work in women treated for breast cancer: a questionnaire-based study.

BACKGROUND: Breast cancer survivors often perceive reduced work ability upon returning to work. OBJECTIVES: To identify predictors of perceived reduced work ability following return to work among women treated for breast cancer and to describe workplace interventions and support after returning to work. METHODS: A questionnaire was sent to 18-65 years-old women (no. 1578) treated for breast cancer and residing in the catchment area of the Bologna Local Health Authority between 2010 and 2012. The study population was identified through a Hospital Discharge Database. The questionnaires included items about personal characteristics, cancer and work-related factors, perceived work ability and the return to work process. A multivariable logistic regression analysis was performed to identify predictors of reduced work ability upon returning to work. RESULTS: Among the 841 respondents, 503 questionnaires were evaluable. In the study, 43.5% of the respondents reported reduced work ability with respect to the pre-diagnosis period. Reduced work ability was more common in non-cohabiting (OR=1.81, 95%CI 1.10-2.98) than in cohabiting/married women, and after mastectomy (OR=2.77, 95%CI 1.26-6.11) than after breast-conserving surgery. Office staff/sales assistants and managers were less likely to report reduced work ability (OR=0.51, 95%CI 0.30-0.88 and OR=0.21, 95%CI 0.06-0.76, respectively) than labourers. Women who perceived reduced work ability reported more frequently adjustment of work assignments, consultation of an occupational physician, insufficient support from employers and colleagues and discrimination. CONCLUSIONS: Reduced work ability is commonly perceived among women who return to work after treatment for breast cancer. Occupational physicians and general practitioners should be aware of a wide range of factors influencing this perception in order to facilitate a successful return to work.

Rapid and green discrimination of bovine milk according to fat content, thermal treatment, brand and manufacturer via colloidal fingerprinting.

Addressing food safety and detecting food fraud while fulfilling greenness requisites for analysis is a challenging but necessary task. The use of sustainable techniques, with limited pretreatment, non-toxic chemicals, high throughput results, is recommended. A combination of Field Flow Fractionation (FFF), working in saline carrier and with minimal preprocessing, and chemometrics was for the first time applied to bovine milk grouping. A set of 47 bovine milk samples was analyzed: a single analysis yielded a characteristic multidimensional colloidal dataset, that once processed with multivariate tools allowed simultaneously for different discriminations: fat content, thermal treatment, brand and manufacturing plant. The analytical methodology is fast, green, simple, and inexpensive and could offer great help in the field of quality control and frauds identification. This work represents also the first attempt to identify milk sub-typologies based on colloidal profiles, and the most complete study concerning multivariate analysis of FFF fingerprint.

Native characterization and QC profiling of human amniotic mesenchymal stromal cell vesicular fractions for secretome-based therapy.

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension. This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities. This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.

Follow-up of early breast cancer in a public health system: A 2024 AIGOM consensus project.

Breast cancer stands as the most frequently diagnosed cancer and the primary cause of cancer-related mortality among women worldwide, including Italy. With the increasing number of survivors, many are enrolled in regular follow-up programs. However, adherence to recommendations from scientific societies (such as ASCO, ESMO, AIOM) for breast cancer follow-up management varies in daily clinical practice across different cancer centers, potentially resulting in unequal management and escalating costs. To address these concerns, the Italian Association of Multidisciplinary Oncology Groups (AIGOM) orchestrated a Consensus on early Breast Cancer follow-up utilizing the Estimate-Talk-Estimate methodology. Following the identification of 18 Items and 38 statements by a select Board, 46 out of 54 (85.1%) experts comprising a multidisciplinary and multiprofessional panel expressed their degree of consensus (Expert Panel). The Expert Panel underscores the potential for the multidisciplinary team to tailor follow-up intensity based on the individual risk of recurrence. In selected cases, the general practitioner may be recommended as the clinical lead for breast cancer follow-up, both after completion of adjuvant treatment and at early initiation of endocrine therapy in low-risk patients. Throughout follow-up, and alongside oncologic surveillance, the expert panel advises osteometabolic, cardiologic, and gynecologic surveillance for the early detection and management of early and late treatment toxicities. Moreover, preserving quality of life is emphasized, with provisions for psycho-oncologic support and encouragement to adopt protective lifestyle behaviors.

Liposomes characterization for market approval as pharmaceutical products: Analytical methods, guidelines and standardized protocols.

Liposomes are nano-sized lipid-based vesicles widely studied for their drug delivery capabilities. Compared to standard carries they exhibit better properties such as improved site-targeting and drug release, protection of drugs from degradation and clearance, and lower toxic side effects. At present, scientific literature is rich of studies regarding liposomes-based systems, while 14 types of liposomal products have been authorized to the market by EMA and FDA and many others have been approved by national agencies. Although the interest in nanodevices and nanomedicine has steadily increased in the last two decades the development of documentation regulating and standardizing all the phases of their development and quality control still suffers from major inadequacy due to the intrinsic complexity of nano-systems characterization. Many generic documents (Type 1) discussing guidelines for the study of nano-systems (lipidic and not) have been proposed while there is a lack of robust and standardized methods (Type 2 documents). As a result, a widespread of different techniques, approaches and methodologies are being used, generating results of variable quality and hard to compare with each other. Additionally, such documents are often subject to updates and rewriting further complicating the topic. Within this context the aim of this work is focused on bridging the gap in liposome characterization: the most recent standardized methodologies suitable for liposomes characterization are here reported (with the corresponding Type 2 documents) and revised in a short and pragmatical way focused on providing the reader with a practical background of the state of the art. In particular, this paper will put the accent on the methodologies developed to evaluate the main critical quality attributes (CQAs) necessary for liposomes market approval.

The dual nature of biomimetic melanin.

Melanin-inspired nanomaterials offer unique photophysical, electronic and radical scavenging properties that are widely explored for health and environmental preservation, or energy conversion and storage. The incorporation of functional melanin building blocks in more complex nanostructures or surfaces is typically achieved via a bottom-up approach starting from a molecular precursor, in most cases dopamine. Here we demonstrate that indeed, the oxidative polymerization of dopamine, for the synthesis of melanin-like polydopamine (PDA), leads to the simultaneous formation of more than one nanosized species with different compositions, morphologies and properties. In particular, a low-density polymeric structure and dense nanoparticles (NP) are simultaneously formed. The two populations could be separated and analyzed in real time using a chromatographic technique free of any stationary phase (flow field fractionation, FFF). The results following the synthesis of melanin-like PDA showed that the NP are formed only during the first 6 hours as a result of a supramolecular self-assembly-driven polymerization, while the formation of the polymer continues for about 36 hours. The two populations were also separated and characterized using TEM, UV-vis absorption spectroscopy, fluorescence and light scattering spectroscopy, DLS, FTIR, ζ-potential measurements, gel electrophoresis and pH titrations. Interestingly, very different properties between the two populations were observed: in particular the polymer contains a higher number of catechol units (8 mmol g-1 -OH) with respect to the NP (1 mmol g-1 -OH) and presents a much higher antioxidant activity. The attenuation of light by NP is more efficient than that by the polymer especially in the Vis-NIR region. Moreover, while the NP scatter light with an efficiency up to 27% they are not fluorescent, and the polymer does not scatter light but shows an excitation wavelength-dependent fluorescence typical of multi-fluorophoric uncoupled systems.

Synthesis Monitoring, Characterization and Cleanup of Ag-Polydopamine Nanoparticles Used as Antibacterial Agents with Field-Flow Fractionation.

Advances in nanotechnology have opened up new horizons in nanomedicine through the synthesis of new composite nanomaterials able to tackle the growing drug resistance in bacterial strains. Among these, nanosilver antimicrobials sow promise for use in the treatment of bacterial infections. The use of polydopamine (PDA) as a biocompatible carrier for nanosilver is appealing; however, the synthesis and functionalization steps used to obtain Ag-PDA nanoparticles (NPs) are complex and require time-consuming cleanup processes. Post-synthesis treatment can also hinder the stability and applicability of the material, and dry, offline characterization is time-consuming and unrepresentative of real conditions. The optimization of Ag-PDA preparation and purification together with well-defined characterization are fundamental goals for the safe development of these new nanomaterials. In this paper, we show the use of field-flow fractionation with multi-angle light scattering and spectrophotometric detection to improve the synthesis and quality control of the production of Ag-PDA NPs. An ad hoc method was able to monitor particle growth in a TLC-like fashion; characterize the species obtained; and provide purified, isolated Ag-PDA nanoparticles, which proved to be biologically active as antibacterial agents, while achieving a short analysis time and being based on the use of green, cost-effective carriers such as water.

Native Study of the Behaviour of Magnetite Nanoparticles for Hyperthermia Treatment during the Initial Moments of Intravenous Administration.

Magnetic nanoparticles (MNPs) present outstanding properties making them suitable as therapeutic agents for hyperthermia treatments. Since the main safety concerns of MNPs are represented by their inherent instability in a biological medium, strategies to both achieve long-term stability and monitor hazardous MNP degradation are needed. We combined a dynamic approach relying on flow field flow fractionation (FFF)-multidetection with conventional techniques to explore frame-by-frame changes of MNPs injected in simulated biological medium, hypothesize the interaction mechanism they are subject to when surrounded by a saline, protein-rich environment, and understand their behaviour at the most critical point of intravenous administration. In the first moments of MNPs administration in the patient, MNPs change their surrounding from a favorable to an unfavorable medium, i.e., a complex biological fluid such as blood; the particles evolve from a synthetic identity to a biological identity, a transition that needs to be carefully monitored. The dynamic approach presented herein represents an optimal alternative to conventional batch techniques that can monitor only size, shape, surface charge, and aggregation phenomena as an averaged information, given that they cannot resolve different populations present in the sample and cannot give accurate information about the evolution or temporary instability of MNPs. The designed FFF method equipped with a multidetection system enabled the separation of the particle populations providing selective information on their morphological evolution and on nanoparticle-proteins interaction in the very first steps of infusion. Results showed that in a dynamic biological setting and following interaction with serum albumin, PP-MNPs retain their colloidal properties, supporting their safety profile for intravenous administration.

Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment.

(1) Background: Hydrogen sulfide (H2S) is a widely recognized gasotransmitter, with key roles in physiological and pathological processes. The accurate quantification of H2S and reactive sulfur species (RSS) may hold important implications for the diagnosis and prognosis of diseases. However, H2S species quantification in biological matrices is still a challenge. Among the sulfide detection methods, monobromobimane (MBB) derivatization coupled with reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most reported. However, it is characterized by a complex preparation and time-consuming process, which may alter the actual H2S level; moreover, a quantitative validation has still not been described. (2) Methods: We developed and validated an improved analytical protocol for the MBB RP-HPLC method. MBB concentration, temperature and sample handling were optimized, and the calibration method was validated using leave-one-out cross-validation and tested in a clinical setting. (3) Results: The method shows high sensitivity and allows the quantification of H2S species, with a limit of detection of 0.5 µM. Finally, it can be successfully applied in measurements of H2S levels in the serum of patients subjected to inhalation with vapors rich in H2S. (4) Conclusions: These data demonstrate that the proposed method is precise and reliable for measuring H2S species in biological matrices and can be used to provide key insights into the etiopathogenesis of several diseases and sulfur-based treatments.

First Case of Typhoid Fever due to Extensively Drug-resistant Salmonella enterica serovar Typhi in Italy.

Typhoid fever is a potentially severe and occasionally life-threatening bacteraemic illness caused by Salmonella enterica serovar Typhi (S. Typhi). In Pakistan, an outbreak of extensively drug-resistant (XDR) S. Typhi cases began in November 2016. We report on a five-year-old boy who contracted enteric fever while travelling in Pakistan and was diagnosed after returning to Italy in September 2019. Blood culture isolated Salmonella enterica serovar Typhi that was XDR to all first-line antibiotics, including ceftriaxone and fluoroquinolones. Empiric therapy was switched to meropenem, and the patient recovered completely. Whole-genome sequencing showed that this isolate was of haplotype H58. The XDR S. Typhi clone encoded a chromosomally located resistance region and harbored a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum ß-lactamase and the qnrS fluoroquinolone resistance gene. This is the first case of typhoid fever due to XDR S. Typhi detected in Italy and one of the first paediatric cases reported outside Pakistan, highlighting the need to be vigilant for future cases. While new vaccines against typhoid are in development, clinicians should consider adapting their empiric approach for patients returning from regions at risk of XDR S. Typhi outbreak with typhoid symptoms.

Risk factors for colonization with carbapenemase-producing Klebsiella pneumoniae in hospital: a matched case-control study.

The present case-control study aimed to identify the main factors associated with colonization with carbapenemase-producing Klebsiella pneumoniae (CPKP) in acute care facilities. Administration of carbapenems (odds ratio [OR], 3.67; 95% confidence interval [CI], 1.37-9.83) and other antibiotics (OR, 2.83; 95% CI, 1.10-7.31) during the hospital stay was significantly associated with outcome. The design of this study, with concurrent selection of controls and matching by hospital ward and date of screening, provides a more accurate estimation of relative risks for CPKP colonization than previous investigations.

A validation study of the WHO analgesic ladder: a two-step vs three-step strategy.

GOALS OF WORK: The aims of the present study were to verify whether an innovative therapeutic strategy for the treatment of mild-moderate chronic cancer pain, passing directly from step I to step III of the WHO analgesic ladder, is more effective than the traditional three-step strategy and to evaluate the tolerability and therapeutic index in both strategies. METHODS: Patients aged 18 years or older with multiple viscera or bone metastases or with locally advanced disease were randomized. Pain intensity was assessed using a 0-10 numerical rating scale based on four questions selected from the validated Italian version of the Brief Pain Inventory. Treatment-specific variables and other symptoms were recorded at baseline up to a maximum follow-up of 90 days per patient. RESULTS: Fifty-four patients were randomized onto the study, and pain intensity was assessed over a period of 2,649 days. The innovative treatment presented a statistically significant advantage over the traditional strategy in terms of the percentage of days with worst pain > or =5 (22.8 vs 28.6%, p < 0.001) and > or =7 (8.6 vs 11.2%, p = 0.023). Grades 3 and 4 anorexia and constipation were more frequently reported in the innovative strategy arm, although prophylactic laxative therapy was used less in this setting. CONCLUSIONS: Our preliminary data would seem to suggest that a direct move to the third step of the WHO analgesic ladder is feasible and could reduce some pain scores but also requires careful management of side effects.