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In the last one-hundred years, the exponential expansion of wine making has artificialized the agricultural landscape as well as its microbial diversity, spreading human selected Saccharomyces cerevisiae strains. Evidence showed that social wasps can harbor a significant fraction of the yeast phenotypic diversity of a given area of wine production, allowing different strains to overwinter and mate in their gut. The integrity of the wasp-yeast ecological interaction is of paramount importance to maintain the resilience of microbial populations associated to wine aromatic profiles. In a field experiment, we verified whether Polistes dominula wasps, reared in laboratory and fed with a traceable S. cerevisiae strain, could be a useful tool to drive the controlled yeast dispersion directly on grapes. The demonstration of the biotechnological potential of social insects in organic wine farming lays the foundations for multiple applications including maintenance of microbial biodiversity and rewilding vineyards through the introduction of wasp associated microbiomes. [ABSTRACT FROM AUTHOR]

The quality of life of patients affected by Parkinson's disease is improved by medications containing levodopa and carbidopa, restoring the dopamine concentration in the brain. Accordingly, the affordable quality control of such pharmaceuticals is very important. Here is reported the simple and inexpensive colorimetric quantification of carbidopa in anti-Parkinson drugs by the selective condensation reaction between the hydrazine group from carbidopa and the formyl functional group of selected aldehydes in acidified hydroalcoholic solution. An optical assay was developed by using indole-3-carbaldehyde (I3A) giving a yellow aldazine in EtOH:H2O 1:1 (λmax~415 nm) at 70 °C for 4 h, as confirmed by LC-MS analysis. A filter-based plate reader was used for colorimetric data acquisition, providing superior results in terms of analytical performances for I3A, with a sensitivity ~50 L g−1 and LOD ~0.1 mg L−1 in comparison to a previous study based on vanillin, giving, for the same figures of merit values, about 13 L g−1 and 0.2–0.3 mg L−1, respectively. The calibration curves for the standard solution and drugs were almost superimposable, therefore excluding interference from the excipients and additives, with very good reproducibility (avRSD% 2–4%) within the linear dynamic range (10 mg L−1–50 mg L−1). [ABSTRACT FROM AUTHOR]

Background: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. Methods: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. Results: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. Conclusions: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts. [ABSTRACT FROM AUTHOR]

• High rate plants/m2 promoted higher phenolic content in some ancient wheat varieties. • Campestanyl and sitostanyl ferulates were the main steryl ferulates in wheat. • Steryl ferulates were proposed as markers for cereals differentiation. • Ancient whole-wheat pasta showed 50 % higher steryl ferulates than modern one. A big challenge for our century concerns discovering new foods, including re-discovering ancient wheat. Phenolic and steryl ferulates composition of Tuscan and Campania wheat samples was evaluated by HPLC-DAD-MS/MS. Cinnamic acids and flavonoids were expressed as ferulic acid and schaftoside equivalents, respectively. Agronomic conditions with a higher rate of plants/m2 promoted higher amounts of phenols in most of the Tuscan varieties. The ancient varieties Gentil Rosso (1137 μg/mg dry weight) and Carosella 2 (854 μg/mg dry weight) showed the highest phenols amount, determined by HPLC-DAD. Steryl ferulates were expressed as ferulic acid equivalents. Campestanyl and sitostanyl ferulates were found as main steryl ferulates compounds in wheat, differently from rice, millet and sorghum; 10 steryl ferulates and 2 caffeoyl phytosterols were tentatively identified. Total steryl ferulates in Tuscan and Campania wheat samples ranged 37.6–62.3 μg/g dry weight and 14.4–56.6 μg/g dry weight, respectively. Ancient whole wheat pasta showed 50 % higher steryl ferulates with respect to modern whole wheat pasta. The study compared for the first time the content of γ-oryzanol derivatives in ancient and modern wheat varieties. [ABSTRACT FROM AUTHOR]

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard's reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples. [ABSTRACT FROM AUTHOR]

MDPHP is a synthetic cathinone (SC) belonging to α-pyrrolidinophenone derivatives. It is a central nervous system stimulant and may induce hallucinations, paranoia, tachycardia, hypertension, chest pain, and rhabdomyolysis. In literature, a few cases of intoxication have been reported. In the present study, 17 cases of MDPHP intake were described including the analytical findings and clinical manifestations. MDPHP was quantified by liquid chromatography–tandem mass spectrometry in blood (range 1.26–73.30 ng/mL) and urine (range 19.31–8769.64 ng/mL) samples. In three cases the presence of α-PHP was observed. In one case, MDPHP was the only detected substance. Concomitant use of MDPHP with other substances, particularly psychostimulants, was common and it was difficult to describe the peculiar clinical characteristics of this SC. Most of the symptoms overlapped those expected, some of them were unusual and all of them particularly severe thus inducing the research of NPS in laboratory tests. We demonstrated the presence of psychiatric, neurological, and respiratory symptoms, as well as the possible presence of rhabdomyolysis and cardiotoxicity associated with the use of MDPHP. ED admissions were also more frequent in patients with addiction problems. In some cases, MDPHP intake required intensive supportive care. A multidisciplinary approach, including specialist consultation, is recommended for patients showing challenging features. Moreover, we demonstrated that the adoption of advanced analytical techniques, i.e., liquid chromatography–tandem mass spectrometry, is necessary to detect these molecules. Further studies are needed to understand MDPHP intake patterns and associated symptoms. It is essential to raise awareness in addiction treatment centers and among potential users, especially young people, and chemsex addicted. • 17 MDPHP intoxication cases were described in this paper. • Clinical symptoms and concentrations were reported. • MDPHP causes dystonia, psychomotor agitation, delirium, and sensory-perception alterations. [ABSTRACT FROM AUTHOR]

• A LC-MS/MS method for the assay of nintedanib and its impurities was developed. • Method development was carried out by Analytical Quality by Design approach. • A 35//16 symmetric matrix was used in the screening phase. • Box-Behnken Design allowed the contour plots to be obtained. • Method operable design region was defined by drawing probability map. Nintedanib (NIN) is a tyrosine kinase inhibitor recently approved for the treatment of idiopathic pulmonary fibrosis. As a new drug, no monograph is available so far in official compendia. A liquid chromatography-tandem mass spectrometry method is presented for the simultaneous determination of NIN and its seven potential impurities. The risk-based approach of Analytical Quality by Design was applied in method development. The critical method parameters (CMPs) were the type of organic solvent in the mobile phase, formic acid percentage, column flow rate, oven temperature, gradient slope of organic eluent. The critical method attributes (CMAs) were selected as analysis time and selectivity between the main compound NIN and the adjacent peaks. Design of Experiments methodology was effectively employed for establishing the relationship between the CMPs and the CMAs. In the scouting step, a Restek Ultra AQ C18 (100 × 2.1 mm, 2.7 µm) core-shell column was selected, and then the effects of different levels of the five CMPs on the CMAs were evaluated by means of a 35//16 symmetric screening matrix. A Box-Behnken Design made it possible to obtain detailed maps of predicted CMAs throughout the investigated experimental domain, pointing out the presence of interaction and quadratic effects. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations, performing a risk analysis and drawing risk of failure maps, which were used to visualize and define the method operable design region (MODR) with a probability π ≥ 90%. The final working conditions (enclosing the MODR interval) were as follows: methanol as organic solvent; formic acid percentage, 0.15% v/v; flow rate, 0.40 mL min−1 (0.37–0.43 mL min−1); oven temperature, 40 °C (38–40°C); gradient slope of organic eluent, 14.00% eluent B min−1 (12.85–15.15% eluent B min−1). The resulting analysis time was about 10 min. Validation was carried out according to International Council for Harmonisation guidelines and the optimized method was applied to the analysis of NIN soft capsules for quality control purposes. [ABSTRACT FROM AUTHOR]

CCDC78 was identified as a novel candidate gene for autosomal dominant centronuclear myopathy-4 (CNM4) approximately ten years ago. However, to date, only one family has been described, and the function of CCDC78 remains unclear. Here, we analyze for the first time a family harboring a CCDC78 nonsense mutation to better understand the role of CCDC78 in muscle., Methods: We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined CCDC78 transcripts and protein using WB in CCDC78 -mutated muscle tissue; these analyses were also performed on muscle, lymphocytes, and fibroblasts from healthy subjects. Subsequently, we conducted RT-qPCR and transcriptome profiling through RNA-seq to evaluate changes in gene expression associated with CCDC78 dysfunction in muscle. Lastly, coimmunoprecipitation (Co-Ip) assays and mass spectrometry (LC-MS/MS) studies were carried out on extracted muscle proteins from both healthy and mutated subjects., Results: The histopathological features in muscle showed novel histological hallmarks, which included areas of dilated and swollen sarcoplasmic reticulum (SR). We provided evidence of nonsense-mediated mRNA decay (NMD), identified the presence of novel CCDC78 transcripts in muscle and lymphocytes, and identified 1035 muscular differentially expressed genes, including several involved in the SR. Through the Co-Ip assays and LC-MS/MS studies, we demonstrated that CCDC78 interacts with two key SR proteins: SERCA1 and CASQ1. We also observed interactions with MYH1, ACTN2, and ACTA1., Conclusions: Our findings provide insight, for the first time, into the interactors and possible role of CCDC78 in skeletal muscle, locating the protein in the SR. Furthermore, our data expand on the phenotype previously associated with CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4.

Aliphatic chlorinated hydrocarbons, notably tetrachloroethylene (also known as perchloroethylene [PCE]), are persistent, mobile, and toxic (PMT) and/or very persistent, mobile, and toxic (vPMT) groundwater pollutants, often exceeding safe drinking water thresholds. The present study delves into the groundwater risk assessment of PCE with a novel focus on the sensitivity of stygobitic species-organisms uniquely adapted to groundwater environments. Through a comparative analysis of the subchronic effects of PCE on the locomotion behavior of two copepod species, the stygobitic Moraria sp. and the nonstygobitic Bryocamptus zschokkei, we highlighted the inadequacy of the current European predicted-no-effect concentration of PCE for groundwater ecosystems. Our findings indicate significant behavioral impairments in both species at a concentration (32 ng/L PCE) well below the threshold deemed safe, suggesting that the current European guidelines for groundwater risk assessment may not adequately protect the unique biodiversity of groundwater habitats. Importantly, B. zschokkei demonstrated sensitivity to PCE comparable to or greater than that of the target stygobitic species, suggesting its utility as a substitute species in groundwater risk assessment. The present study adds to the limited research on the ecotoxicological sensitivity of groundwater species to PMT/vPMT chemicals and highlights the need for refined groundwater risk-assessment methodologies that consider the susceptibilities of stygobitic species. Environ Toxicol Chem 2024;00:1-13. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Celiac disease (CD) is an autoimmune enteropathy resulting from an interaction between diet, genome, and immunity. Although many patients respond to a gluten-free diet, in a substantive number of individuals, the intestinal injury persists. Thus, other factors might amplify the ongoing inflammation. Candida albicans is a commensal fungus that is well adapted to the intestinal life. However, specific conditions increase Candida pathogenicity. The hypothesis that Candida may be a trigger in CD has been proposed after the observation of similarity between a fungal wall component and two CD-related gliadin T-cell epitopes. However, despite being implicated in intestinal disorders, Candida may also protect against immune pathologies highlighting a more intriguing role in the gut. Herein, we postulated that a state of chronic inflammation associated with microbial dysbiosis and leaky gut are favorable conditions that promote C. albicans pathogenicity eventually contributing to CD pathology via a mast cells (MC)-IL-9 axis. However, the restoration of immune and microbial homeostasis promotes a beneficial C. albicans -MC cross-talk favoring the attenuation of CD pathology to alleviate CD pathology and symptoms., (© 2024 Renga et al.)

Abstract: CPX-351, a liposomal combination of cytarabine plus daunorubicin, has been approved for the treatment of adults with newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes, because it improves survival and outcome of patients who received hematopoietic stem cell transplant compared with the continuous infusion of cytarabine plus daunorubicin (referred to as "7 + 3" combination). Because gut dysbiosis occurring in patients with AML during induction chemotherapy heavily affects the subsequent phases of therapy, we have assessed whether the superior activity of CPX-351 vs "7 + 3" combination in the real-life setting implicates an action on and by the intestinal microbiota. To this purpose, we have evaluated the impact of CPX-351 and "7 + 3" combination on mucosal barrier function, gut microbial composition and function, and antifungal colonization resistance in preclinical models of intestinal damage in vitro and in vivo and fecal microbiota transplantation. We found that CPX-351, at variance with "7 + 3" combination, protected from gut dysbiosis, mucosal damage, and gut morbidity while increasing antifungal resistance. Mechanistically, the protective effect of CPX-351 occurred through pathways involving both the host and the intestinal microbiota, namely via the activation of the aryl hydrocarbon receptor-interleukin-22 (IL-22)-IL-10 host pathway and the production of immunomodulatory metabolites by anaerobes. This study reveals how the gut microbiota may contribute to the good safety profile, with a low infection-related mortality, of CPX-351 and highlights how a better understanding of the host-microbiota dialogue may contribute to pave the way for precision medicine in AML., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases., (© 2024. The Author(s).)

Background: The recent Coronavirus Disease 2019 (COVID-19) pandemic has dramatically exposed our gap in understanding the pathogenesis of airborne infections. Within such a context, it is increasingly clear that the nasal cavity represents a critical checkpoint not only in the initial colonization phase but also in shaping any infectious sequelae . This is particularly relevant to COVID-19 in that the nasal cavity is characterized by high-level expression of the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) receptor, Angiotensin-Converting Enzyme 2 (ACE2), all along the respiratory tract. As part of the nasal mucosa, commensal microbes harbored by the nasal cavity likely are far more than just innocent bystanders in the interaction between SARS-CoV-2 and the local microenvironment. Yet the role of the qualitative composition of the nasal microbiome is unclear, as is its function, whether protective or not., Methods: In this study, individuals undergoing SARS-CoV-2 molecular testing at the Hospital of Perugia (Italy) were recruited, with their residual material from the nasopharyngeal swabs being collected for microbiome composition analysis and short-chain fatty acid (SCFA) measurements (by 16S rRNA sequencing and gas chromatography-mass spectrometry), respectively., Results: After stratification by age, gender, and viral load, the composition of the nasopharyngeal microbiome appeared to be influenced by age and gender, and SARS-CoV-2 infection further determined compositional changes. Notwithstanding this variability, a restricted analysis of female subjects-once SARS-CoV-2-infected-unraveled a shared expansion of Lachnospirales-Lachnospiraceae, irrespective of the viral load and age. This was associated with a reduction in the branched SCFA isobutanoic acid, as well as in the SCFAs with longer chains., Conclusions: Our results indicate that the nasopharyngeal microbiome is influenced by age, gender, and viral load, with consistent patterns of microbiome changes being present across specific groups. This may help in designing a personalized medicine approach in COVID-19 patients with specific patterns of nasal microbial communities., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Background: Atrial fibrillation (AF) is associated with chronic inflammation, a hallmark of ageing process. The aim of this study was to determine interleukin-6 (IL-6)-associated variables, also exploring acylcarnitines, expression of mitochondrial abnormalities., Methods: We evaluated 22 controls and 50 patients with persistent AF. IL-6 and acylcarnitines were measured with ELISA kits and mass spectrometry techniques., Results: IL-6 concentration (mean: 3.9 ± 3.1 pg/mL) was lower in controls and increased in AF patients, especially with heart failure. The CHA 2 DS 2 -VASc, the MMSE and the SPPB scores were 3.8 ± 1.6, 28 ± 2 and 9.4 ± 2.1. Thirteen acylcanitines correlated with IL-6. At multivariable analysis, IL-6 was directly associated with C4-OH-a short-chain acylcarnitine, fibrinogen and alanine aminotransferase values, and with hyperuricemia. An inverse association existed with calcium concentration and SPPB score., Conclusions: In older AF patients, IL-6 correlated with acylcarnitines and lower physical performance. Alterations in energy production, reduced physical function and inflammation could contribute to frailty development., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Shotgun proteomics is a very sensitive bottom-up approach used to study complex mixtures of proteins through a combination of high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS). This technique has been successfully applied to the study of olfactory appendixes of insects and other arthropods. Since extracting soluble proteins from tissues and processing them for shotgun proteomics is quite straightforward, the technique has proved to be very suitable for studying soluble olfactory proteins, such as odorant binding proteins (OBPs), chemosensory proteins (CSPs) and Niemann-Pick type 2 proteins. Here, we describe the main principles and methods at the basis of shotgun proteomics, including its use to quantify proteins through label-free quantification. We also provide operational protocols for the experimental workflow steps, i.e., insect dissection, protein extraction, protein enzymatic digestion, sample purification, HPLC-MS analysis, search of mass spectral data against polypeptide databases and first steps in the analysis of the search results., (© 2020 Elsevier Inc. All rights reserved.)

Capparis spinosa L. is a perennial plant typical of the Mediterranean flora and a multipurpose plant used for curing various human ailments. Quaternary ammonium compounds (QACs), as constituents of Capparaceae, play important roles in protecting against abiotic stress. Aim of this work was to determine QACs in root and leaves of caper from two proveniences. The presence of stachydrine, choline, glycine betaine and homo-stachydrine has been confirmed by high resolution MS, while 1 H NMR was applied to quantify the main QACs in the aqueous extracts. Stachydrine was quantified at 20.2 mg/g and 32.3 mg/g on dry leaves from South of Italy and Saudi Arabia, respectively, while a minor content was in dry roots (from 10.4 to 12.5 mg/g). Choline was considerably lower both in leaves and roots (from 0.3 to 1.2 mg/g). To our knowledge, this is the first report on the determination of QACs both in root and leaves of C. spinosa.

Unlabelled: Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effect in epithelial cancers, including hepatocellular carcinoma (HCC). The effective anticancer properties and the underlying molecular mechanisms of these drugs in vivo remain unclear. In addition, the primary biological target of TZD, the ligand-dependent transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), is up-regulated in HCC and seems to provide tumor-promoting responses. The aim of our study was to evaluate whether chronic administration of TZD may affect hepatic carcinogenesis in vivo in relation to PPARgamma expression and activity. The effect of TZD oral administration for 26 weeks was tested on tumor formation in PPARgamma-expressing and PPARgamma-deficient mouse models of hepatic carcinogenesis. Proteomic analysis was performed in freshly isolated hepatocytes by differential in gel electrophoresis and mass spectrometry analysis. Identified TZD targets were confirmed in cultured PPARgamma-deficient hepatocytes. TZD administration in hepatitis B virus (HBV)-transgenic mice (TgN[Alb1HBV]44Bri) reduced tumor incidence in the liver, inhibiting hepatocyte proliferation and increasing apoptosis. PPARgamma deletion in hepatocytes of HBV-transgenic mice (Tg[HBV]CreKOgamma) did not modify hepatic carcinogenesis but increased the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as a TZD target in PPARgamma-deficient hepatocytes. TZD inhibited NPM expression at protein and messenger RNA levels and decreased NPM promoter activity. TZD inhibition of NPM was associated with the induction of p53 phosphorylation and p21 expression., Conclusion: These findings suggest that chronic administration of TZD has anticancer activity in the liver via inhibition of NPM expression and indicate that these drugs might be useful for HCC chemoprevention and treatment.

Background. Clodronate is used in high bone resorption diseases. Its action was defined as "cytotoxic" based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As "in vitro" bioeffects of clodronate are reversible, this compound should not be defined as "cytotoxic".Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2)side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a β-γ-methylene analog of ATP that induces inhibition of the ADP/ATP translocase.Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiative modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex "matrix metalloproteinase 9/tissue inhibitor of metalloproteinase" (MMP9/TIMP1) by RT-PCR analysis based on "TaqMan" technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5'-[ β - γ -dichloromethylene] triphosphate (AppCCl(2)p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney "U" test was conducted for statistical analysis.Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl(2)p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage.Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl(2)p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP.