Cancer cells show increased glucose uptake and lactate secretion due to mitochondrial respiratory dysfunction and hypoxia, leading to extracellular acidification of the tumor microenvironment (TME) and intracellular alkalinization. This metabolic reprogramming promotes malignant phenotypes, including enhanced invasion, metastasis, multidrug resistance, and immune evasion. Therefore, real-time monitoring of intra- and extracellular pH dynamics is essential to understand tumor progression and to evaluate therapeutic strategies. Here, we report the use of a pH-sensitive bioluminescent color-tuning biosensor, derived from the firefly Amydetes vivianii luciferase (AmyLuc), to ratiometrically estimate intracellular and extracellular pH changes associated with metabolic alterations consistent with the Warburg effect in human colorectal adenocarcinoma cells (Caco-2). The ratio of bioluminescence emission intensities at 593 nm (pH 6.0) and 548 nm (pH 8.0) was used to establish a calibration curve for accurate pH determination. Analysis of the green/red emission ratios showed that the treatments with the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 50 μM) and the respiratory chain inhibitor antimycin A (50 μM) induced a sustained intracellular acidification (pH ∼6.3), whereas the extracellular environment showed a gradual alkalinization toward near-physiological pH (∼7.1), consistent with buffering effects of the medium. This intracellular acidification is consistent with metabolic alterations and intracellular proton accumulation caused by the transition from mitochondrial respiration to cytoplasmic anaerobic glycolysis. The results highlight the suitability of AmyLuc as a sensitive color-tuning bioluminescent pH biosensor for real-time monitoring of pH dynamics in cancer cells under metabolic stress and therapeutic interventions.

The replacement of enzyme labels such as horseradish peroxidase (HRP) with noble metal nanoparticles represents a promising approach in biosensing. Ultrasmall platinum nanoparticles (PtNPs) exhibit remarkable catalytic activity, chemical stability, and low production costs, making them attractive nanozyme candidates also in the field of highly sensitive chemiluminescence (CL) detection. However, their CL performances strongly depend on particle size, shape, composition, and concentration, often resulting in limited reproducibility and robustness. These physicochemical parameters also affect biomolecular labeling efficiency, including conjugation with proteins and antibodies, which is critical for consistent biosensor performance. To enable the development of robust biosensors integrating nanozymes as active sensing elements, rigorous nanoparticle quality control and efficient isolation of their bioconjugates are key steps. In this work, we report the first application of hollow-fiber flow field-flow fractionation (HF5) for the characterization and isolation of bioconjugated ultrasmall PtNPs designed for CL bioassays. Shape-controlled pyramidal PtNPs were synthesized in aqueous media via a rapid, scalable, and surfactant-free route exploiting sodium citrate as a shape-directing agent. PtNPs were characterized to define optimal nanoparticle-to-protein ratios, ensuring high conjugate stability. HF5 was then employed to selectively isolate standardized conjugated PtNPs, which were directly employed for CL signal generation. Finally, a proof-of-concept study demonstrated the use of conjugated IgG-PtNPs isolated through HF5 for the quantitative detection of a primary antibody in a paper-based CL bioassay, achieving a limit of detection suitable for diagnostic applications. Thus, HF5-mediated streamlining of probe synthesis and isolation offered a very promising answer to unmet needs in biosensor development.

Colorectal cancer (CRC) onset is closely linked to dysregulated Notch signaling, particularly involving receptors like Notch1 and Notch3, and ligands such as Jagged1 (Jag1), which are overexpressed in aggressive subtypes. To address the need for a quantitative assessment of Notch expression in clinical settings, we developed a luminescent recombinant probe (Jag1-Fluc) by fusing the extracellular domain of a high-affinity Jag1 mutant with red-emitting firefly luciferase. The Jag1-Fluc probe can bind to Notch, thus correlating the intensity of emitted light with the concentration of Notch in the sample. In cell-free assays, Jag1-Fluc demonstrated a linear luminescent signal across different concentrations (detection limit: 0.20 ± 0.03 μg/mL). When applied to living and fixed human colorectal cancer cells, the probe showed a dose-dependent light emission (5-50 μg/mL), confirming its ability to bind endogenous Notch receptors. Competitive binding assays using soluble human Jag1 chimera demonstrated concentration-dependent signal inhibition (IC₅₀ = 0.55 ± 0.06 μg/mL), validating probe specificity. Notably, in a small cohort of human biopsies, Jag1-Fluc enabled stratification of Notch expression, with luminescence intensity progressively increasing from hyperplastic polyps to low- and high-grade adenomas. These findings reinforce prior evidence linking Jag1 overexpression to CRC progression. The assay's simplicity and sensitivity highlight its potential for early CRC screening, paving the way for personalized medicine by enabling tailored therapeutic monitoring and strategies based on Notch signaling profiles. This approach potentially enhances treatment efficacy and reduces side effects, representing a valuable tool for precision oncology.

Chemiluminescence (CL) and bioluminescence (BL) are both fascinating natural phenomena involving the emission of light by a chemical reaction without the need for an excitation source. Both CL and BL have been used extensively in research and technology development and have become valuable tools in fields ranging from chemistry to medicine and beyond. Specifically, they offer unique advantages in the design of highly sensitive and specific detection probes for biosensors. In the CL field, novel probe designs, integration of nanomaterials, and synthetic strategies have led to enhanced sensitivity, selectivity, and versatility in biosensor applications. This in combination with portable microfluidic technologies has facilitated the detection and quantification of biomolecules, metabolites, pathogens, and environmental pollutants with high accuracy and low cost. Similarly, the exploration of bioluminescent proteins has led to the engineering of genetically encoded bioluminescent probes enabling the development of biosensors, molecular diagnostics methods, and real-time imaging and detection within living systems. Further, the development of bioluminescent reporter assays and biosensors has shown application in high-throughput screening, drug discovery, and biological mechanistic studies. The integration of BL and CL technologies into point-of-care devices has found applications in medical diagnostics, environmental monitoring, and food safety. This review highlights recent advancements in both CL and BL technologies, mainly focusing on their implications in biosensor development.

The fight against coronavirus disease 2019 (COVID-19) continues. Since the pandemic’s onset, several biomarkers have been proposed to assess the diagnosis and prognosis of this disease. This research aimed to identify potential disease severity biomarkers in serum samples of patients with COVID-19 during the disease course. Data were collected using untargeted and targeted mass spectrometry methods. The results were interpreted by performing univariate and multivariate analyses. Important metabolite classes were identified by qualitative untargeted metabolomics in 15 serum samples from survivors of COVID-19. Quantitative targeted metabolomics on a larger patient cohort including 15 non-survivors confirmed serum 3-sulfate bile acids (i.e. GLCA-3S) were significantly increased in non-survivors compared to survivors during the early disease stage (p-value < 0.0001). Notably, it was associated with a higher risk of mortality (odds ratio of 26). A principal component analysis showed the ability to discriminate between survivors and non-survivors using the BA concentrations. Furthermore, increased BA-S is highly correlated with known parameters altered in severe clinical conditions.

The release of hazardous chemicals into aquatic environments has long been a known problem, but its full impact has only recently been realized. This study presents a validated liquid chromatography-mass spectrometry (HPLC-MS/MS) method for detecting pharmaceutical and pesticide residues in mussels (Mytilus galloprovincialis). An innovative MS-compatible extraction method was developed and validated, demonstrating successful recovery rates for analytes at three different concentration levels (25-95%). The method detected the target analytes at ng/g concentrations with high accuracy (-7% to 11%) and low relative standard deviation (<10%) for both intra-day and inter-day analyses. After validation, the method was applied to mussel samples collected from a commercial farm near Senigallia, Adriatic Sea, detecting different contaminants in the range of 2-40 ng/g (dry weight). The study provides a valuable tool for investigating the potential threats posed by diverse contaminant classes with high annual tonnage, including analytes with known persistence and/or illegal status.

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.

Bile acids (BAs), endogenous acidic steroids synthetized from cholesterol in the liver, play a key role in the gut–liver axis physiopathology, including in hepatotoxicity, intestinal inflammatory processes, and cholesterol homeostasis. Faecal Oxo-BAs, relatively stable intermediates of oxidation/epimerization reactions of the BA hydroxyls, could be relevant to investigating the crosstalk in the liver–gut axis and the relationship between diseases and alterations in microbiota composition. A paucity of information currently exists on faecal BA profiles in dogs with and without chronic inflammatory enteropathy (CIE). Comprehensive assessment of 31 molecules among faecal BAs and related microbiota metabolites was conducted with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Odds ratios (ORs) for associations of BAs with CIE were estimated using logistic regression. Principal component analysis was performed to find differences between the control and pathological dogs. Higher levels of primary BAs and muricholic acids, and lower levels of secondary BAs were found in pathological dogs. Higher concentrations in faecal oxo-metabolites were associated with the absence of CIE (OR < 1). This study shows a marked difference in faecal BA profiles between dogs with and without CIE. Further research will be needed to better understand the role of oxo-BAs and muricholic acids in CIE dogs.

The Farnesoid X Receptor (FXR) is the master regulator of Bile Acids (BA) homeostasis orchestrating their synthesis, transport and metabolism. Disruption of BA regulation has been linked to gut-liver axis diseases such as colorectal cancer (CRC). In this study, firstly we examined the role of constitutive activation of intestinal FXR in CRC; then we pre-clinically investigated the therapeutic potential of a diet enriched with a synthetic FXR agonist in two models of CRC (chemically-induced and genetic models). We demonstrated that mice with intestinal constitutive FXR activation are protected from AOM/DSS-induced CRC with a significant reduction of tumor number compared to controls. Furthermore, we evaluated the role of chemical FXR agonism in a DSS model of colitis in wild type (WT) and FXRnull mice. WT mice administered with the FXR activating diet showed less morphological alterations and decreased inflammatory infiltrates compared to controls. The FXR activating diet also protected WT mice from AOM/DSS-induced CRC by reducing tumors' number and size. Finally, we proved that the FXR activating diet prevented spontaneous CRC in APCMin/+ mice via an FXR-dependent modulation of BA homeostasis. Our results demonstrate that intestinal FXR activation prevented both inflammation- and genetically-driven colorectal tumorigenesis by modulating BA pool size and composition. This could open new avenues for the therapeutic management of intestinal inflammation and tumorigenesis.

The standard method for estimating the chemical oxygen demand (COD) of water bodies uses dichromate as the main oxidant, a chemical agent whose use has been restricted in the European Union since 2017. This method is hazardous, time-consuming, and burdensome to adapt to on-site measurements. As an alternative and following the current trends of sustainable and green chemistry, a method using the less toxic reagent sodium persulfate as the oxidizing agent has been developed. In this method an excess of persulfate, activated through heating in an alkaline solution, oxidizes the chemically degradable organic fraction through a 2-step radical mechanism. The remaining persulfate is evaluated by chemiluminescence (CL) using luminol and a portable charge-coupled device (CCD) camera. The method provided quantitative recoveries and a sample throughput of >60 samples h(-1). It was validated in river water samples by comparison of COD estimations with the standard dichromate method (R = 0.973, p < 0.05) and with a UV-Vis permanganate-based method (R = 0.9998, p < 0.05), the latter being also used for drinking waters. The proposed method is a sustainable and green alternative to the previous used methods. Overall, the method using activated persulfate is suitable for use as COD quantitation/screening tool in surface waters. Considering that its main components are portable, it can be ultimately adapted for in situ analysis at the point of need.

Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH2 and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with luc genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.

Bones and teeth are biological archives, but their structure and composition are subjected to alteration overtime due to biological and chemical degradation postmortem , influenced by burial environment and conditions. Nevertheless, organic fraction preservation is mandatory for several archeometric analyses and applications. The mutual protection between biomineral and organic fractions in bones and teeth may lead to a limited diagenetic alteration, promoting a better conservation of the organic fraction. However, the correlation between elemental variations and the presence of organic materials (e.g., collagen) in the same specimen is still unclear. To fill this gap, chemiluminescent (CL) immunochemical imaging analysis has been applied for the first time for collagen localization. Then, Laser Ablation–Inductively Coupled Plasma–Mass Spectrometry (LA–ICP–MS) and CL imaging were combined to investigate the correlation between elemental (i.e., REE, U, Sr, Ba) and collagen distribution. Teeth and bones from various archeological contexts, chronological periods, and characterized by different collagen content were analyzed. Immunochemical analysis revealed a heterogeneous distribution of collagen, especially in highly degraded samples. Subsequently, LA–ICP–MS showed a correlation between the presence of uranium and rare earth elements and areas with low amount of collagen. The innovative integration between the two methods permitted to clarify the mutual relation between elemental variation and collagen preservation overtime, thus contributing to unravel the effects of diagenetic alteration in bones and teeth.

N-substituted acridine-containing 1,2-dioxetanes have been recently proposed as thermochemiluminescence (TCL) universal labels for bioanalytical applications. The TCL properties of these compounds markedly depend on the nature of substituents of the acridine ring. In the attempt to obtain new TCL probes with improved properties and stability, the push-pull approach was adopted, in which both electron withdrawing and electron donating groups are present in the acridine moiety. The results have been useful to better understand the role of the different decorations at the acridine fluorophore in modulating the photophysical properties and the activation parameters of 1,2-dioxetane labels. Moreover, the great versatility and innovation of these molecules make them extremely attractive for bioanalytical applications, in particular as labels for immune- and gene-probe assays.

There is a growing interest in the named “acidic sterolbiome” and in the genetic potential of the gut microbiome (GM) to modify bile acid (BA) structure. Indeed, the qualitative composition of BAs in feces correlates with the bowel microorganisms and their collective genetic material. GM is responsible for the production of BA metabolites, such as secondary and oxo-BAs. The specific BA profiles, as microbiome-host co-metabolic products, could be useful to investigate the GM-host interaction in animals under physiological conditions, as well as in specific diseases. In this context, we developed and validated an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for the simultaneous analysis of up to 21 oxo-BAs and their 9 metabolic precursors. Chromatographic separation was achieved in 7 min with adequate analytical performance in terms of selectivity, sensitivity (LOQ from 0.05 to 0.1 µg/mL), accuracy (bias% < 5%), precision (CV% < 5%) and matrix effect (ME% < 10%). A fast solvent extraction protocol has been fine-tuned, achieving recoveries > 90%. In parallel, the gut microbiota assessment in farming animals was evaluated by 16S rRNA next-generation sequencing, and the correlation with the BA composition was performed by multivariate analysis, allowing to reconstruct species-specific associations between the BA profile and specific GM components.

BACKGROUND:Acute kidney injury (AKI) is common in advanced liver cirrhosis, a consequence of reduced kidney perfusion due to splanchnic arterial vasodilation and intrarenal vasoconstriction. It clinically manifests as hepatorenal syndrome type 1, type 2, or as acute tubular necrosis. Beyond hemodynamic factors, an additional mechanism may be hypothesized to explain the renal dysfunction during liver cirrhosis. Recent evidence suggest that such mechanisms may be closely related to obstructive jaundice.METHODS:Given the not completely elucidated role of bile acids in kidney tissue damage, this study developed a rat model of AKI with liver cirrhosis induction by carbon tetrachloride (CCl4) inhalation for 12 weeks. Histological analyses of renal and liver biopsies were performed at sacrifice. Organic anion tubular transporter distribution and apoptosis in kidney cells were analyzed by immunohistochemistry. Circulating and urinary markers of inflammation and tubular injury were assayed in 21 treated rats over time (1, 2, 4, 8, and 12 weeks of CCl4 administration) and 5 controls.RESULTS:No renal histopathological alterations were found at sacrifice. Comparing treated rats with controls, organic anion transporters were differentially expressed and localized. High serum bile acid values were detected in cirrhotic animals, while caspase-3 staining was negative in both groups. Increased levels of serum inflammatory and urinary tubular injury biomarkers were observed during cirrhosis progression, with a peak after 4 and 8 weeks of treatment.CONCLUSIONS:These findings suggest possible adaptive tubular mechanisms for bile acid transporters in response to cirrhosis-induced AKI.

Background: Currently, there is no clinically established method to predict critically ill Coronavirus Disease -19 (COVID-19) patients. Few prognostic biomarkers are available to predict severe outcomes of patients and then differentiate customized therapies.Methods: We performed an untargeted metabolomics study in 15 serum samples of COVID-19 - survivor patients to establish potential severity biomarkers. Then, a targeted metabolomics study was performed on a larger patient cohort, including 15 non-survivors, at different time points (T1-T2-T3) during the disease course. The results were interpreted by performing univariate and multivariate analyses.Finding: Serum glycolithocholic acid 3- sulfate (GLCA-3S) was increased with a significant difference between survivors and non-survivors in the early stage of the illness (T1 p-value < 0 ·0001), highly correlated with mortality (p-value <0·001). The specificity and sensitivity values of GLCA-3S for predicting early clinical diagnosis were 80·0% and 85·7%, respectively. PLS-DA model was able to identify with high selectivity the most critical patients in the early stages of COVID-19.Interpretation: This study identifies sulfated bile acids (BA-S) as biomarkers with altered serum levels in a cohort of COVID-19 patients. Moreover, increased BA-S are highly correlated with the well-known parameters altered in the worse clinical conditions of COVID-19 patients. GLCA-3S was identified as the main biomarker correlated with mortality in the early stage of the illness.Funding Information: We acknowledge support from the Ministry of Education, University and Research of Italy: PRIN 2017 (Project 2017Y2PAB8) and the EU Horizon 2020 program INFRAIA project Epic-XS (Project 823839).Declaration of Interests: No competing interests.Ethics Approval Statement: The studies involving human participants were reviewed and approved by Comitato Etico Area Vasta Emilia Centro, Bologna, Italy. The patients/participants provided their written informed consent to participate in this study. In case of unconsciousness, the informed consent was signed by the next of kin or legal authorized representative.

Introduction:Most patients with metabolic syndrome present non-alcoholic fatty liver disease (NAFD).High consumption of fruit and vegetable has been related to a lower incidence of chronic diseases.Of note, among vegetables, brassicaceae family is at the first place.Therapeutic strategies to treat inflammatory disease as well as cancer are evolving and includes biologically active phytocompounds.A sulfur-containing glucosinolate (GLS) secondary plant compounds exclusively present in Brassicaceae-and their breakdown products including isothiocyanates (NCS) have shown to possess important anti-inflammatory and anti-carcinogenic property.The aim of this work was the evaluation of the effects of daily consumption of E. sativa defatted seed meal enriched bakery products on glucose and lipid metabolism and on systemic markers of inflammation. Material and Methods:After an 8-week run-in period, patients were asked for 4 weeks to maintain their usual diet replacing 150 gr. of bakery products with the same number of enriched bakery product.Fasting glucose and insulin, HOMA-r, total cholesterol, HDL, LDL, triglyceride, ALT, Gamma-GT, high sensitivity C reactive protein, Interleukin-6, Interleukin-8 and TNF-α were compared before and after the study period.Results: HOMA-r showed a statistically significant reduction after 4 weeks period as well as cholesterol ratio 3.8 ± 1.0 vs. 3.6 ± 1.0 (P = 0.03).Among markers of inflammation, high sensitivity CRP showed a statistically significant reduction (-36.8%)from 1.4 (0.4 -10.4) to 0.9 (0.4 -8.7) mg/dl (P = 0.02) as well as TNF-α 8.0 ± 3.0 vs. 7.1 ± 2.7 pg/ml (-11.3%;P < 0.001).Despite a mild ALT reduction (-7.0%),Gamma-GT showed a -21.0%decrease from 20.0 ± 6.6 to 15.8 ± 6.4 U/L (P < 0.001).IL-6 and IL-8 were substantially unmodified. Conclusion:as cracker can improve glucose and lipids metabolism in parallel with a significant improvement of serum Bakery products enriched with Eruca sativa Mill defatted seed meal markers of inflammation and therefore being of interest for patients affected by inflammatory diseases.

In recent years, there has been an increasing demand for predictive and sensitive in vitro tools for drug discovery. Split complementation assays have the potential to enlarge the arsenal of in vitro tools for compound screening, with most of them relying on well-established reporter gene assays. In particular, ligand-induced complementation of split luciferases is emerging as a suitable approach for monitoring protein–protein interactions. We hereby report an intracellular nanosensor for the screening of compounds with androgenic activity based on a split NanoLuc reporter. We also confirm the suitability of using 3D spheroids of Human Embryonic Kidney (HEK-293) cells for upgrading the 2D cell-based assay. A limit of detection of 4 pM and a half maximal effective concentration (EC50) of 1.7 ± 0.3 nM were obtained for testosterone with HEK293 spheroids. This genetically encoded nanosensor also represents a new tool for real time imaging of the activation state of the androgen receptor, thus being suitable for analysing molecules with androgenic activity, including new drugs or endocrine disrupting molecules.

Bile acid researchers throughout the world were saddened to learn of the death of Alan F. Hofmann, Professor Emeritus of Medicine at the University of California, San Diego (UCSD), on September 7, 2021. Alan was an internationally renowned basic scientist and clinical investigator, whose more than 60 years of research shaped and transformed our understanding of the chemistry, biology, and physiology of bile acids in health and disease. His long and remarkable career was marked by seminal scientific discoveries and by his service as an ambassador for the field of “cholanology” (the science of bile acids) to attract, educate, and inspire new generations of physicians and scientists. Alan Hofmann was born on May 17, 1931 to Joseph and Nellie Hofmann and grew up in Baltimore, MD. He graduated from Baltimore City College for High School and was awarded a full scholarship to Johns Hopkins University. Besides his academic subjects, Alan Hofmann also played bass violin in the orchestra, sousaphone in the band, and sang glee club. He graduated with academic honors in 3 years and remained at Johns Hopkins for Medical School, having been awarded a full academic scholarship. After completing his internship at Columbia-Presbyterian, and a stint as a clinical associate at the National Institutes of Health, Alan Hofmann's career was at a crossroads. At the National Institutes of Health, he became interested in the problem of fat digestion and believed that the most knowledgeable person on the subject at that time (∼1959) was the lipid biochemist Bengt Borgström in Sweden. While waiting to hear about his fellowship application to study with Dr Borgström, Alan Hofmann left the National Institutes of Health to join the National Symphony as an accompanying physician on its Pan American tour. It was in Peru that Alan Hofmann learned his fellowship would be funded and he was soon off to the University of Lund. Over the next 3 years, Drs Hofmann and Borgström conducted their groundbreaking work to elucidate the physicochemical properties of bile acids in solution and their relationship to fat absorption, as well as showing that the ileum is the major site of conjugated bile acid absorption in humans. For his thesis describing this work, Alan Hofmann received an MD (equivalent of a PhD) from the University of Lund in 1965, and it was with a twinkle in his eye that Alan Hofmann traveled back to Lund in 2015, where on May 29, he was celebrated as a “Jubeldoctor” (Jubilee doctor, honorary doctorate awarded 50 years after the original doctorate). For these discoveries regarding lipid digestion and absorption, the American Gastroenterological Association (AGA) recognized Alan Hofmann in 1970 with a Distinguished Achievement Award, and Drs Hofmann and Borgström were awarded the William Beaumont Prize in 1979. After returning from Sweden, Alan Hofmann joined Rockefeller University in New York, where he worked with Scott Grundy to study the impact of bile acids on cholesterol metabolism in humans and fostered a productive outside collaboration with Erwin Mosbach to understand the principles underlying calcium-bile acid interactions in bile. In 1966, Alan Hofmann moved to the Mayo Clinic in Rochester, where he served as the codirector of clinical and basic gastrointestinal research until 1977. Alan Hofmann thrived at the Mayo. It was there that Alan Hofmann, together with a remarkable group of collaborators and talented fellows that included Sydney Phillips, Hanns Fromm, Leslie Schoenfield, Rudy Dazinger, Rainer Poley, Johnson Thistle, Nicholas LaRusso, and Tim Northfield, made major advancements in our understanding of bile acid malabsorption, the kinetics of daily bile secretion and enterohepatic cycling in humans, and oral bile acid (chenodeoxycholic acid) therapy for dissolution of cholesterol gallstones. Since 1977, Alan Hofmann has been a Professor in the Department of Medicine at the UCSD. There, Alan Hofmann and collaborators worked to define the relationship between bile acid structure and micelle formation and to further our understanding of the physiology and pharmacokinetic principles underlying the enterohepatic circulation of bile acids. Particularly fruitful was his work with Karol Mysels at UCSD, a renown physical chemist and expert on micelles and surface tension, and Aldo Roda, a chemist and visiting scholar from the University of Bologna. Together, they advanced our understanding of the relationship between bile acid structure and self-aggregation for micelle formation in solution. This work (1Roda A. Hofmann A.F. Mysels K. The influence of bile salt structure on self-association in aqueous solutions.J. Biol. Chem. 1983; 258: 6362-6370Abstract Full Text PDF PubMed Google Scholar) remains highly cited and opened new perspectives on the design of semisynthetic bile acid analogs as drugs. Alan Hofmann was always excited about advances in bioanalytical methods for measuring bile acids and helped develop the first radioimmunoassay for primary bile acids and worked with Gerard van Berge Henegouwen on the use of gas-liquid chromatography while at the Mayo Clinic. This work continued at UCSD with the development of a simple and sensitive bioluminescent assay for serum primary bile acids (with Aldo Roda), a precursor to biosensors and point-of-care testing, and application of state-of-the-art mass spectrometry-based methods to the study of bile acids and bile acid metabolites. Together with his close colleague, Lee Hagey (http://bile.ucsd.edu/), Alan Hofmann launched an ambitious program to understand the remarkable diversity of chemical structures in the three great bile salt classes, C27 bile alcohols, C27 bile acids, and C24 bile acids, building upon the work of the late Geoffrey Haslewood, Professor of Biochemistry at Guy's Hospital Medical School in London. Much of this work appeared in the Journal of Lipid Research, including analysis of the biliary bile salt analysis of 677 vertebrate species with Matthew Krasowski in 2010 (2Hofmann A.F. Hagey L.R. Krasowski M. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51: 226-246Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In 1994, Alan Hofmann was awarded the Friedenwald Medal from the AGA for his many discoveries over the previous 35 years. When asked how he may wind down his career, Alan Hofmann was quoted as saying: “I want to know why we have these molecules. We started off believing that bile acids helped us absorb lipids. We clarified that. Next, we clarified their role in the liver—how they induce bile flow and lipid secretion. But I am not sure that is the answer.” Over the next 27 years, Alan saw his work and influence on the field foster a bile acid renaissance, with the emergence of new answers to his question and exciting advances in our understanding of bile acids as signaling molecules and regulators of metabolism and the microbiome. During this period and in keeping with his goal of “trying to understand the chemistry and biology of bile acids with the hope of helping patients,” Alan Hofmann and his work directly and indirectly catalyzed the development of new bile acid-related therapeutics. This includes approved therapies such as the second-generation bile acid sequestrant colesevelam, the semisynthetic farnesoid X-receptor agonist obeticholic acid, and ileal bile acid transporter inhibitors odevixibat and maralixibat, and bile acid-based therapies in development such as the bile salt hydrolase-resistant bile acid analog cholylsarcosine as bile acid replacement therapy for short bowel syndrome, 11C-cholylsarcosine for use in positron emission tomography imaging, and the side chain shortened bile acid analog, norursodeoxycholic acid (norucholic acid), for primary sclerosing cholangitis. The story behind obeticholic acid is illustrative of Alan Hofmann's influence. At a party in the early 1980s at UCSD, Alan Hofmann and Aldo Roda met Roberto Pellicciari, a visiting professor in Medicinal Chemist from Perugia. After returning to Italy, Drs Roda and Pellicciari initiated a collaboration to design, synthesize, and study the physicochemical properties of new bile acid analogs. This culminated in the discovery of obeticholic acid (6α-ethyl-chenodeoxycholic acid) by Roberto Pellicciari, who was motivated by Alan Hofmann's encouragement and his prediction of the existence of bile acid receptors. Alan Hofmann will be remembered for his enthusiasm, creativity, and sense of humor. Alan Hofmann generously shared his encyclopedic knowledge of bile acids and was an early mainstay of National and International Bile Acid Meetings. Alan Hofmann co-organized (with Luigi Barbara and Enrico Roda from the University of Bologna) the Biennial Congress on Bile Acid Research at the Italian mountain village of Cortina d'Amprezzo in 1975 and subsequent years, and organized in 1983 the last Kroc Foundation Conference on the Physical Chemistry of Bile in Health and Disease. The Kroc Foundations Conference convened a remarkable group of individuals from the physical and biomedical sciences, and a picture and list of the participants is shown. In 1972, Alan Hofmann together with Gustav Paumgartner launched with Biennial International Bile Acid Meetings, which continue to be supported by the Falk Foundation, and remain an outstanding venue to present advances in bile acid biology, chemistry, and therapeutics. During his career, Alan Hofmann published over 500 original articles and invited contributions as well as 51 reviews. The Journal of Lipid Research remained one of Alan Hofmann's favorite journal to publish his basic research on bile acids, including an authoritative history of key discoveries in bile acid chemistry and biology over the past 8 decades (3Hofmann A.F. Hagey L. Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades.J. Lipid Res. 2014; 55: 1553-1595Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Alan Hofmann also made two short films, edited nine books, coauthored one book, cofounded the Undergraduate Teaching Project of the AGA and published four sets of teaching materials in gastrointestinal physiology and pathophysiology. In addition to the awards mentioned previously, Alan Hofmann has been the recipient of many other honors and honorary degrees. These include an honorary degree in medicine from the Alma Mater Studiorum University of Bologna Italy (1988), the Davenport Medal from the American Physiological Society (1996), the Thannhauser Medal from the Germany Society for Gastroenterology, Digestive and Metabolic Diseases (1996), a Distinguished Achievement Award from the American Association for the Study of Liver Diseases (1997), and Herbert Falk Medal (2010) from the Falk Foundation. Alan Hofmann's legacy is embodied in his lasting impact on the bile acid field, the patients that his findings have helped or will help in the future, and the shared memories of his family and his many friends and admirers from around the world. Alan is survived by Heli Hofmann, his wife of 43 years, two daughters Anthea and Cecelia from his first marriage, two stepdaughters Caroline and Isabel, and five grandchildren. His passing leaves an immeasurable void, and he will be greatly missed.

Food waste is a global problem due to its environmental and economic impact, so there is great demand for the exploitation of new functional applications. The winemaking process leads to an incomplete extraction of high-value compounds, leaving the pomace still rich in polyphenols. This study was aimed at optimising and validating sustainable routes toward the extraction and further valorisation of these polyphenols, particularly for cosmeceutical applications. New formulations based on red grape pomace polyphenols and natural deep eutectic solvents (NaDESs) were here investigated, namely betaine combined with citric acid (BET-CA), urea (BET-U) and ethylene glycol (BET-EG), in which DESs were used both as extracting and carrying agents for polyphenols. The flavonoid profile determined by HPLC-MS/MS analysis showed similar malvidin content (51–56 μg mL−1) in the DES combinations, while BET-CA gave the best permeation performance in Franz cells, so it was further investigated in 3D human keratinocytes (HaCat spheroids) injured with the pro-oxidant agent menadione. BET-CA treatment showed good intracellular antioxidant activity (IC50 0.15 ± 0.02 μg mL−1 in malvidin content) and significantly decreased (p < 0.001) the release of the pro-inflammatory cytokine IL-8, improving cell viability. Thus, BET-CA formulation is worthy of investigation for potential use as a cosmetic ingredient to reduce oxidative stress and inflammation, which are causes of skin aging.