Design and validation of a reporter mouse to study the dynamic regulation of TFEB and TFE3 activity through in vivo imaging techniques.

TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor's validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism.Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

Compliance between Food and Feed Safety: Eight-Year Survey (2013-2021) of Aflatoxin M1 in Raw Milk and Aflatoxin B1 in Feed in Northern Italy.

Aflatoxins (AFs) are fungal metabolites that are found in feed and food. When ruminants eat feed contaminated with aflatoxin B1 (AFB1), it is metabolised and aflatoxin M1 (AFM1) is excreted in the milk. Aflatoxins can result in hepatotoxic, carcinogenic, and immunosuppressive effects. The European Union thus set a low threshold limit (50 ng/L) for presence of AFM1 in milk. This was in view of its possible presence also in dairy products and that quantification of these toxins is mandatory for milk suppliers. In the present study, a total of 95,882 samples of whole raw milk, collected in northern Italy between 2013 and 2021, were evaluated for presence of AFM1 using an ELISA (enzyme-linked immunosorbent assay) method. The study also evaluated the relationship between feed materials collected from the same farms in the same area during the same period (2013-2021) and milk contamination. Only 667 milk samples out of 95,882 samples analysed (0.7%) showed AFM1 values higher than the EU threshold limit of 50 ng/L. A total of 390 samples (0.4%) showed values between 40 and 50 ng/L, thus requiring corrective action despite not surpassing the regulatory threshold. Combining feed contamination and milk contamination data, some feedingstuffs seem to be more effective in defying potential carryover of AFs from feed to milk. Combining the results, it can be concluded that a robust monitoring system that covers both feed, with a special focus on high risk/sentinel matrices, and milk is essential to guarantee high quality and safety standards of dairy products.

Sex-Specific Microglial Responses to Glucocerebrosidase Inhibition: Relevance to GBA1-Linked Parkinson's Disease.

Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson's Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5-2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1.

Increased Sensitivity of Computed Tomography Scan for Neoplastic Tissues Using the Extracellular Vesicle Formulation of the Contrast Agent Iohexol.

Computed tomography (CT) is a diagnostic medical imaging modality commonly used to detect disease and injury. Contrast agents containing iodine, such as iohexol, are frequently used in CT examinations to more clearly differentiate anatomic structures and to detect and characterize abnormalities, including tumors. However, these contrast agents do not have a specific tropism for cancer cells, so the ability to detect tumors is severely limited by the degree of vascularization of the tumor itself. Identifying delivery systems allowing enrichment of contrast agents at the tumor site would increase the sensitivity of detection of tumors and metastases, potentially in organs that are normally inaccessible to contrast agents, such as the CNS. Recent work from our laboratory has identified cancer patient-derived extracellular vesicles (PDEVs) as effective delivery vehicles for targeting diagnostic drugs to patients' tumors. Based on this premise, we explored the possibility of introducing iohexol into PDEVs for targeted delivery to neoplastic tissue. Here, we provide preclinical proof-of-principle for the tumor-targeting ability of iohexol-loaded PDEVs, which resulted in an impressive accumulation of the contrast agent selectively into the neoplastic tissue, significantly improving the ability of the contrast agent to delineate tumor boundaries.

An Eight-Year Survey on Aflatoxin B1 Indicates High Feed Safety in Animal Feed and Forages in Northern Italy.

Aflatoxins (AFs) remain the main concern for the agricultural and dairy industries due to their effects on the performances and quality of livestock production. Aflatoxins are always unavoidable and should be monitored. The objective of this paper is to bring to light a significant volume of data on AF contamination in several animal feed ingredients in Northern Italy. The Regional Breeders Association of Lombardy has been conducting a survey program to monitor mycotoxin contamination in animal feeds, and in this paper, we present data relating to AFB1 contamination. In most cases (95%), the concentrations were low enough to ensure compliance with the European Union's (EU's) maximum admitted levels for animal feed ingredients. However, the data show a high variability in AF contamination between different matrices and, within the same matrix, a high variability year over year. High levels of AFs were detected in maize and cotton, especially in the central part of the second decade of this century, i.e., 2015-2018, which has shown a higher risk of AF contamination in feed materials in Northern Italy. Variability due to climate change and the international commodity market affect future prospects to predict the presence of AFs. Supplier monitoring and control and reduced buying of contaminated raw materials, as well as performing analyses of each batch, help reduce AF spread.

Dietary essential amino acids restore liver metabolism in ovariectomized mice via hepatic estrogen receptor α.

In female mammals, the cessation of ovarian functions is associated with significant metabolic alterations, weight gain, and increased susceptibility to a number of pathologies associated with ageing. The molecular mechanisms triggering these systemic events are unknown because most tissues are responsive to lowered circulating sex steroids. As it has been demonstrated that isoform alpha of the estrogen receptor (ERα) may be activated by both estrogens and amino acids, we test the metabolic effects of a diet enriched in specific amino acids in ovariectomized (OVX) mice. This diet is able to block the OVX-induced weight gain and fat deposition in the liver. The use of liver-specific ERα KO mice demonstrates that the hepatic ERα, through the control of liver lipid metabolism, has a key role in the systemic response to OVX. The study suggests that the liver ERα might be a valuable target for dietary treatments for the post-menopause.

Transplantation of autologous extracellular vesicles for cancer-specific targeting.

Nano- and microsized extracellular vesicles (EVs) are naturally occurring cargo-bearing packages of regulatory macromolecules, and recent studies are increasingly showing that EVs are responsible for physiological intercellular communication. Nanoparticles encapsulating anti-tumor theranostics represent an attractive "exosome-interfering" strategy for cancer therapy. Methods: Herein, by labeling plasma-derived EVs with indocyanine green (ICG) and following their biodistribution by in vivo and ex vivo imaging, we demonstrate the existence of nanoparticles with a highly selective cancer tropism in the blood of colorectal cancer (CRC) patients but not in that of healthy volunteers. Results: In CRC patient-derived xenograft (PDX) mouse models, we show that transplanted EVs recognize tumors from the cognate nanoparticle-generating individual, suggesting the theranostic potential of autologous EVs encapsulating tumor-interfering molecules. In large canine breeds bearing spontaneous malignant skin and breast tumors, the same autologous EV transplantation protocol shows comparable safety and efficacy profiles. Conclusions: Our data show the existence of an untapped resource of intercellular communication present in the blood of cancer patients, which represents an efficient and highly biocompatible way to deliver molecules directly to the tumor with great precision. The novel EV-interfering approach proposed by our study may become a new research direction in the complex interplay of modern personalized cancer therapy.

Differential Somatic Cell Count as a Marker for Changes of Milk Composition in Cows with Very Low Somatic Cell Count.

The recent availability of a high-throughput milk analyzer performing a partial differential somatic cell count (DSCC) opened new opportunities in investigations on bovine udder health. This analyzer has a potential limitation on the accuracy of measurements when the somatic cell count (SCC) is below 50,000 cells/mL, values characterizing a good proportion of lactating cows in many herds. We obtained data for cows below this threshold, assessed the repeatability of these measurements and investigated the relationship between DSCC and udder health, milk composition and yield. Overall, 3022 cow milk test records performed on a Fossomatic™ 7/DC (Foss A/S, Hillerød, Denmark) were considered; 901 of them had an SCC ≤ 50,000 cells/mL. These latter samples were analyzed by qPCR to identify the presence of bacteria. Overall, 20.75% of the samples (187) were positive. However, the health status did not have any significant association with DSCC. The analysis of the association of DSCC on milk fat, protein and casein showed a significant decrease in their proportions as the DSCC increased, whereas it was not observed for milk yield and lactose. Therefore, DSCC in very low SCC cows may be suggested as a marker to identify early changes in milk composition.

Inhibition of SIRT1 deacetylase and p53 activation uncouples the anti-inflammatory and chemopreventive actions of NSAIDs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application. METHODS: Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs. RESULTS: Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival. CONCLUSION: Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs.

Systemic Administration and Targeted Delivery of Immunogenic Oncolytic Adenovirus Encapsulated in Extracellular Vesicles for Cancer Therapies.

Oncolytic viruses (OV) are engineered to infect, replicate in and kill cancer cells. Currently, the OV therapeutic approach is mainly restricted to neoplasia amenable to direct local administration of viral particles, while the possibility of a systemic delivery of cancer-tropic viruses would extend the OV application to the treatment of metastatic neoplasia. Herein, we applied in vivo/ex vivo imaging to demonstrate that cancer tropism is achieved when OV are encapsulated inside extracellular vesicles (EV) administered intravenously (i.v.), but not when injected intraperitoneally (i.p.). Moreover, we show that the therapeutic procedure adopted does not alter the immunomodulatory properties of the viruses.

Sex-Specific Features of Microglia from Adult Mice.

Sex has a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological diseases, and the fact that estrogens have anti-inflammatory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differences in the transcriptome of adult male and female microglia, possibly originating from perinatal exposure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cerebral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms underscoring a sexual bias in the susceptibility to brain diseases.

In vivo imaging of early signs of dopaminergic neuronal death in an animal model of Parkinson's disease.

The Parkinson's disease (PD) evolves over an extended period of time with the onset occurring long before clinical signs begin to manifest. Characterization of the molecular events underlying the PD onset is instrumental for the development of diagnostic markers and preventive treatments, progress in this field is hindered by technical limitations. We applied an imaging approach to demonstrate the activation of Nrf2 transcription factor as a hallmark of neurodegeneration in neurotoxin-driven models of PD. In dopaminergic SK-N-BE neuroblastoma cells, Nrf2 activation was detected in cells committed to die as proven by time lapse microscopy; in the substantia nigra pars compacta area of the mouse brain, the Nrf2 activation preceded dopaminergic neurodegeneration as demonstrated by in vivo and ex vivo optical imaging, a finding confirmed by co-localization experiments carried out by immunohistochemistry. Collectively, our results identify the Nrf2 signaling as an early marker of neurodegeneration, anticipating dopaminergic neurodegeneration and motor deficits.

PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress.

Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson's disease (PD). PINK1 loss is associated with mitochondrial Ca2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP+-MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca2+ transport..

Novel Locally Active Estrogens Accelerate Cutaneous Wound Healing-Part 2.

Estrogen deprivation is associated with delayed healing, while estrogen replacement therapy (ERT) accelerates acute wound healing and protects against development of chronic wounds. However, current estrogenic molecules have undesired systemic effects, thus the aim of our studies is to generate new molecules for topic administration that are devoid of systemic effects. Following a preliminary study, the new 17ß-estradiol derivatives 1 were synthesized. The estrogenic activity of these novel compounds was evaluated in vitro using the cell line ERE-Luc B17 stably transfected with an ERE-Luc reporter. Among the 17ß-estradiol derivatives synthesized, compounds 1e and 1f showed the highest transactivation potency and were therefore selected for the study of their systemic estrogenic activity. The study of these compounds in the ERE-Luc mouse model demonstrated that both compounds lack systemic effects when administered in the wound area. Furthermore, wound-healing experiments showed that 1e displays a significant regenerative and anti-inflammatory activity. It is therefore confirmed that this class of compounds are suitable for topical administration and have a clear beneficial effect on wound healing.

Identification of novel loci for the generation of reporter mice.

Deciphering the etiology of complex pathologies at molecular level requires longitudinal studies encompassing multiple biochemical pathways (apoptosis, proliferation, inflammation, oxidative stress). In vivo imaging of current reporter animals enabled the spatio-temporal analysis of specific molecular events, however, the lack of a multiplicity of loci for the generalized and regulated expression of the integrated transgenes hampers the creation of systems for the simultaneous analysis of more than a biochemical pathways at the time. We here developed and tested an in vivo-based methodology for the identification of multiple insertional loci suitable for the generation of reliable reporter mice. The validity of the methodology was tested with the generation of novel mice useful to report on inflammation and oxidative stress.

AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells.

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression.

Estrogen accelerates the resolution of inflammation in macrophagic cells.

Although 17ß-estradiol (E2) anti-inflammatory activity has been well described, very little is known about the effects of this hormone on the resolution phase of the inflammatory process. Here, we identified a previously unreported ERα-mediated effect of E2 on the inflammatory machinery. The study showed that the activation of the intracellular estrogen receptor shortens the LPS-induced pro-inflammatory phase and, by influencing the intrinsic and extrinsic programs, triggers the resolution of inflammation in RAW 264.7 cells. Through the regulation of the SOCS3 and STAT3 signaling pathways, E2 facilitates the progression of the inflammatory process toward the IL10-dependent "acquired deactivation" phenotype, which is responsible for tissue remodeling and the restoration of homeostatic conditions. The present study may provide an explanation for increased susceptibility to chronic inflammatory diseases in women after menopause, and it suggests novel anti-inflammatory treatments for such disorders.

In vivo imaging of cell proliferation for a dynamic, whole body, analysis of undesired drug effects.

Noninvasive in vivo imaging offers a novel approach to preclinical studies opening the possibility of investigating biological events in the spatiotemporal dimension (eg, in any district of the body in time). Toxicological analysis may benefit from this novel approach through precise identification of the time and the target organs of toxicity manifestations, and assessment of the reversibility of toxic insults. The current limitation for routine application of this technology is the lack of appropriate surrogate markers for imaging toxicological events. Here, we demonstrate that in vivo imaging of a proliferation marker is capable of measuring the reduction of cell proliferation due to genotoxic/apoptotic agents, γ rays or antineoplastic drugs, or the increased proliferation associated with the inflammatory and regenerative reactions occurring after a toxic insult. A number of tools are currently available for imaging proliferation in preclinical and clinical settings, however our data provide a novel way to translate the evidence of toxic effects obtained in preclinical animal studies, by the direct, noninvasive measure of dividing cells in humans.

Reporter mice for the study of long-term effects of drugs and toxic compounds.

Noninvasive, imaging-based methodologies provide for the first time the possibility to spatio-temporally investigate physiopathological events and long-term effects of drug administration of exposure to environmental and alimentary toxic compounds. Hence, this novel methodology could enable us to measure the dynamics of specific molecular pathways in live animals. In the last few years, animals, particularly mice, were genetically modified to respond to a specific stimulus with the production of proteins, named "reporters," that are easily detected and quantitated by in vivo and ex vivo imaging. These "reporter mice" are gradually being applied to the pharmaco-toxicological research. In the generation of a reporter mouse useful for pharmaco-toxicological studies several elements need to be considered in the selection of the reporter system: the half-life of proteins should be compatible with the necessities of the study to assess the onset and the termination of the stimulus of interest, in all tissues the response should be proportional to the given stimulus, and the imaging modalities requested for reporter measurements should be applicable to high-throughput screening. Bioluminescence-based imaging (BLI) in small animals has the advantage over other modalities that does not require too sophisticated equipment or specifically and highly trained personnel, and furthermore may be carried out at a relative rapidity and low cost; for these reasons several luciferases have been developed for in vivo imaging applications and used in the generation of reporter mice. We here describe a BLI-based reporter mouse created to respond to estrogenic stimuli, which has been applied to the study of female physiopathology as well as for the identification of the effects of selective drugs or toxic compounds present in the environment and in the alimentary chain.

Sexually immature male ERE-Luc reporter mice to assess low dose estrogen-like effects of CdCl2 versus dietary Cd.

CdCl2 salt is widely used in exposure oriented studies, while the biological exposure of Cadmium (Cd) occurs mostly through diet. Hence, we designed a in vivo imaging methodology with sexually immature male ERE-Luc reporter mice to test the estrogen-like (EL) effects of Cd as a natural component in wheat and flax bread based diets (containing 17.57 and 49.22 ug/kg Cd concentrations respectively) and CdCl2 per-oral dose of 1 ug/kg/bw/day. Total exposure of ingested and % bioaccumulation of Cd in selected organs were estimated as 547 ng (4.4%), 776 ng (0.3%) and 2131.8 ng (0.1%) corresponding to CdCl2, wheat and flax bread based diet treatments respectively. Cd from CdCl2 bioaccumulated more readily, despite the exposure of Cd is higher with bread based diets. Longitudinal in vivo imaging did not reveal significant changes in luciferase activity. White adipose tissue (WAT) and prostate were identified as novel target organs of Cd. Indeed, the rest of the observed EL effects, endogenous target gene expression and necropsy findings are not consistent to any particular organ or treatment. This implies that, the observed EL effects due to low doses of Cd (either from CdCl2 or dietary form) occur only as subtle changes at the molecular level, but inadequate to cause significant changes at the anatomo-pathological level during the 21 day exposure period. The study demonstrates the sensitivity of the methodology to assess EL effects of food embedded Cd and underlines the limitations of directly extrapolating the results of suspected chemicals in their pure form to dietary exposure scenarios.