We read with interest the recent study by Gilson and colleagues , “How DoWe evaluated this issue of varying responses using all questions from the most recent quarterly, online, open-book American Board of Preventive Medicine (ABPM) pilot evaluation of a longitudinal assessment program for the maintenance of certification of its clinical informatics diplomates. We evaluated ChatGPT, version 3.5 (OpenAI), and Google Bard (Alphabet Inc) by copying and pasting each of the 12 questions and the corresponding 4-part multiple-choice options into the chatbots’ message boxes on March 30, 2023, and April 1, 2023, respectively. We added a request to provide citations for each question. Both chatbots supplied the option they considered best, with a justification, references, and an explanation as to why each option was either incorrect or inferior to the recommended answer.For ChatGPT, the series of 12 questions was performed 10 times in separate chat sessions to avoid memory effects from a previous search, with each session scored against the answer key provided by the ABPM. The results showed that out of the 12 questions, there were 9 sessions where 8 correct responses were achieved and 1 session where 9 correct responses were achieved. Although 8 questions had perfect (10/10) concordance with the answer key, there were 2 questions with 2 different answers and one with 3 different answers. There was a twelfth question where the same answer was provided for each session that disagreed with the answer key. These scores were at least as good as the average performance of the diplomates participating in the maintenance of certification process , which allows the use of online resources, and likely would have represented a passing score. We also evaluated the experimental ChatGPT, version 4.0, in 5 separate chat sessions, which produced sequential scores of 10, 8, 8, 6, and 7. For Google Bard, the process was performed 9 times, and the most common answer was selected as the best response. The modal responses were correct for 7 out of 12 questions . There were 5 questions for which 2 different answers were provided and 1 question for which all 4 answers were provided as correct answers during different sessions. Google Bard agreed with the ABPM answer key for only 4 questions in all sessions.The questions where the large language models consistently disagreed with the ABPM answer key were either based on low-level evidence or involved an opinion on a “best” approach. As implied by Gilson et al , these d


The significant association between the nadir SpO2 and IPF or non-IPF ILD grouping remained even after adjusting for gender, age, body mass index, lung function, 6MWD, and dyspnea . Conclusion: Even after adjusting for confounding factors, IPF patients had lower nadir SpO2 during 6MWT. Early assessment of exercise desaturation using the 6MWT may be more important in patients with IPF compared with patients with other ILDs.Objective: Interstitial lung disease (ILD) is classified into several disease groups. Among them, idiopathic pulmonary fibrosis (IPF) has higher incidence and poor prognosis; therefore, it is important to characterize specific IPF symptoms. Exercise desaturation is a strong factor related to mortality in patients with ILD. Thus, the purpose of this study was to compare the degree of oxygen desaturation between IPF and other ILD (non-IPF ILD) patients during exercise, using the 6-minute walk test (6MWT). Methods: This retrospective study included 126 stable patients with ILD who underwent 6MWT in our outpatient department. The 6MWT was used to assess desaturation during exercise, 6-minute walk distance (6MWD), and dyspnea at the end of exercise. In addition, patient characteristics and pulmonary function test results were recorded. Results: Study subjects were divided into 51 IPF patients and 75 non-IPF ILD patients. The IPF group had significantly lower nadir oxygen saturation determined by pulse oximetry (SpO Interstitial lung disease (ILD) comprises a group of diverse diseases that damage the lung parenchyma through inflammation and fibrosis. ILD is divided into several disease groups, with different treatment goals and assessment methods1). Idiopathic pulmonary fibrosis (IPF) is the most frequent form of ILD and has a very poor prognosis with a median survival time of 2–3 years from diagnosis2). In contrast, non-IPF ILDs, such as idiopathic nonspecific interstitial pneumonia (NSIP), connective tissue disease-associated ILD, and hypersensitivity pneumonitis, usually have a better prognosis than IPF3). Hence, it is important to characterize the specific symptoms in IPF patients compared with those in non-IPF ILD patients.5). The 6-minute walk test (6MWT) is an inexpensive and simple test that assesses desaturation during exercise and has been shown to be reproducible in patients with ILD7). Desaturation during exercise is a strong risk factor for mortality in ILD patients12) and is also associated with quality of life14). A few studies have assessed the difference in desaturation during exercise between IPF patients and non-IPF ILD patients, but no adjustment was made for some important confounding factors15). Moreover, several studies reported controversial results on whether desaturation during exercise is more severe in patients with IPF17); hence, this relationship remains unclear. Clarifying the differences in desaturation during exercise between patients with IPF and patients with non-IPF ILD will be useful in determining how to evaluate IPF patients and help determine the appropriate treatment strategy.Chronic hypoxemia influences prognosis in patients with ILD. Many patients with ILD present with severe desaturation during exercise even if they do not have hypoxemia at restTherefore, the purpose of this study was to compare the degree of oxygen desaturation in IPF and non-IPF ILD patients during exercise, using the 6MWT.1). Assessment items for the analyses were age, gender, the modified British Medical Research Council (mMRC) scale, comorbidities, treatment, smoking status, 6MWT, and pulmonary function tests.This retrospective study included 195 stable patients with ILD who underwent the 6MWT as part of an examination at our outpatient clinic at Kobe City Medical Center West Hospital (Japan) between April 2016 and March 2020. No respiratory rehabilitation was performed before or after 6MWT because it was performed to assess physiological status of the patients. Among these, patients with long-term oxygen therapy, active coronary artery disease, other severe comorbidities, and missing data were excluded. The classification of ILD, including the diagnosis of IPF, was made using the American Thoracic Society/European Respiratory Society (ATS/ERS) consensus statementThis study was approved by the Institutional Review Board  of Kobe City Medical Center West Hospital with waiver of informed consent because of its retrospective nature .18). Patients were instructed to walk as much as possible in 6 min. Oxygen saturation determined by pulse oximetry (SpO2) during 6MWT was continuously recorded using a pulse oximeter with a finger probe  and 6MWT analysis software . To minimize errors in SpO2 measurement, patients were instructed to hold the finger with the pulse oximeter attached to the front of their chest while walking. SpO2 was also measured at rest for 1 min prior to the test and for 3 min immediately after. Patients were asked to rate their dyspnea every minute during 6MWT, using the modified Borg scale scores by selecting a number from 0 to 10, with 0 being no appreciable dyspnea and 10 being maximal sustained dyspnea19). From the 6MWT, 6-minute walk distance (6MWD), rest SpO2, nadir SpO2, ΔSpO2 (rest SpO2 − nadir SpO2), maximum heart rate, and modified Borg scale scores during 6MWT were obtained.The subjects performed 6MWT on a flat, 25-m walking course according to the ATS statement20). Vital capacity (VC), forced vital capacity (FVC), and forced expiratory volume in 1 s (FEV1) were evaluated. Predicted normal values for the Japanese population were derived from reference values of the Japanese Respiratory Society21) for VC, FVC, and FEV1.All patients were subjected to pulmonary function tests by spirometry  according to the method described in the ATS 1994 update2 and IPF or non-IPF ILD groups. Additionally, multiple regression analysis was performed with the nadir SpO2 as the dependent variable and IPF diagnosis as the independent variable to evaluate the relationship after adjusting for potential covariates. Dummy variables were created with patients with IPF as 0 and non-IPF ILD as 1. These were entered into the regression analysis. Gender, age, body mass index (BMI), chronic obstructive pulmonary disease (COPD) or non-COPD, 6MWD, %VC, and mMRC scale scores were included as confounders because they are assumed to be involved in desaturation23). p-value <0.05 was considered significant.Statistical analysis was performed with EZR Ver. 1.38 . Subjects were categorized into the IPF group or the non-IPF ILD group. Continuous variables were presented as mean ± standard deviation and were compared using unpaired t, Welch’s t, and Mann–Whitney U tests between the two groups. A simple regression analysis was performed to examine the relationship between the nadir SpOAmong 195 patients with ILD who underwent 6MWT, 18 patients were on long-term oxygen therapy and 51 cases had missing data. Therefore, 126 cases were included in the analysis. Fifty-one patients (40%) were diagnosed with IPF and the other 75 patients (60%) had non-IPF ILD. Non-IPF ILD included collagen disease, n = 12 (16.0%); hypersensitivity pneumonitis, n = 10 (13.3%); idiopathic pleuroparenchymal fibroelastosis, n = 6 (8.0%); asbestosis, n = 6 (8.0%); idiopathic NSIP, n = 3 (4.0%); sarcoidosis, n = 2 (2.7%); cryptogenic organizing pneumonia, n = 1 (1.3%); and unclassifiable, n = 35 (46.7%). p = 0.50) and %FVC , between the IPF group and the non-IPF ILD group. In addition, no significant differences were observed in the other pulmonary function measures or patients characteristics between the two groups, with the exception of treatment modalities used for IPF and non-IPF ILD.2 during 6MWT than the non-IPF ILD group . The IPF group also had significantly higher ΔSpO2 during 6MWT than the non-IPF ILD group . In contrast, no significant difference was observed in the dyspnea scale at the end of 6MWT  and the 6MWD  between the two groups.2 over time during the 6MWT.p = 0.02) between nadir SpO2 and IPF or non-IPF ILD grouping (p <0.05) remained even after adjustments for gender, age, BMI, COPD or non-COPD, 6MWD, %VC, and mMRC scale score . Their finding supports our results, but the study did not adjust for any confounding factors. Another study comparing SpO2 during 6MWT between patients with IPF and patients with idiopathic NSIP reported that there was no significant difference5). Although there were only two cases of idiopathic NSIP in the present study, the results could not be simply compared, and it is necessary to classify non-IPF ILD patients in detail and compare the results using 6MWT in the future.The results of this study indicate that patients with IPF tended to have severe desaturation during exercise despite not having hypoxemia at rest. Holland et al. reported that nadir SpO17). Although these results were similar to our findings, 6MWT is a more clinically feasible and simpler exercise stress test than using a bicycle ergometer. In addition, they also did not adjust for confounding factors. Therefore, this study expanded on the results of these two studies and indicated that similar results can also be obtained with 6MWT, which is simple and easy to administer in the clinical settings.We performed the 6MWT to assess desaturation during exercise in patients with ILD. In two previous studies, wherein a bicycle ergometer was employed as an exercise stress test, IPF patients were shown to exhibit more severe desaturation during exercise than non-IPF ILD patientsCO) is more likely to decline earlier compared with %FVC. In fact, it was reported that patients with IPF had lower DLCO than patients with non-IPF ILD24). Ventilation–diffusion inequality and diffusion limitation play an important role in desaturation at rest and during exercise25). A number of studies showed that low DLCO is strongly associated with desaturation during exercise. For example, Du Plessis et al. reported that DLCO was a predictor of nadir SpO2 during 6MWT among ILD patients22). In another study of 300 patients with ILD, a simple regression analysis identified DLCO as the explanatory factor for decreased oxygen saturation during exercise26). Therefore, we speculated that early decline in DLCO in IPF patients could lead to more severe desaturation during exercise. However, this study did not have sufficient data on DLCO, which is a future challenge.More severe desaturation during exercise in patients with IPF might be explained by more severe diffusion limitation. IPF is a progressive disease, and diffusing capacity for carbon monoxide (DL9). Our results indicated that IPF patients have more severe exercise desaturation compared with non-IPF ILD patients. Therefore, early assessment of exercise desaturation using 6MWT may be useful, especially in patients with IPF who do not have hypoxemia at rest. In addition, the 6MWT does not require much time or complex equipment to implement; therefore, the test is easy to perform clinically.Desaturation during exercise is a strong prognostic indicator in patients with IPF28). For that reason, these can be confounding factors. However, in the present study, there was no difference in the maximum pulse rate during the 6MWT, which is related to the circulatory response during exercise, so the difference in circulatory dynamics may not have been significant between IPF and non-IPF ILD patients.Our study has some limitations. First, this was a single-center cross-sectional study and may not represent the general characteristics of IPF patients. Therefore, further multicenter studies with larger samples are needed. Second, we did not evaluate the presence of pulmonary hypertension, right heart pressure, or other circulatory dynamics. It has been reported that patients with pulmonary hypertension present with severe exercise hypoxia2 during 6MWT. Early assessment of exercise desaturation using the 6MWT might be more important in patients with IPF than in patients with non-IPF ILD. Even if the resting SpO2 is good, it may be important to perform 6MWT more aggressively in patients with IPF to detect a decrease in oxygen saturation during the test, which is a prognostic factor at an early stage.The results of this study showed that even after adjusting for confounding factors, IPF patients had lower nadir SpOwww.enago.jp) for the English language review.The authors would like to thank Kiyohiko Kado, Chisato Nagatani, Takashi Kadooka, Shoko Mitani, Takuya Okamoto, Kumi Matumura, Ryoya Fujisaki, Yuya Kawate, Suzuka Higashiyama, Takumi Yamaguchi, Yutaro Oki, Yukari Oki, and Haruko Yamamoto for cooperation in acquisition of data and constructive comments on this paper. The authors would also like to thank Enago (There is no conflict of interest to disclose.


Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1‐dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α‐smooth muscle actin (α‐SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF‐β under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR‐81, a G‐protein‐coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate‐mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR‐81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen‐deficient microenvironments. These molecules serve as proton donors for the oxygen‐dependent electron transport chain and the efficient generation of ATP  is converted to lactate through anaerobic metabolism which generates ATP in a much less efficient manner. Lactate dehydrogenase (LDH), an NADIn some circumstances, best studied in the context of cancer as the “Warburg effect” or “aerobic glycolysis,” pyruvate is converted to lactate even in the presence of sufficient oxygen. Recently, this phenomenon has been characterized as “metabolic reprogramming” in lung fibroblasts stimulated with the pro‐fibrotic cytokine transforming growth factor beta 1 (TGF‐β1)  that is induced in control and IPF fibroblasts under hypoxic conditions in a TGF‐β‐independent manner and regulates lactate‐induced myofibroblast differentiation in control fibroblasts. Taken together, our studies support a novel paradigm in which hypoxic conditions induce IPF fibroblasts to produce extracellular lactate which may then function to directly propagate myofibroblast differentiation in “normal” fibroblasts within the same environment.22.1Lung tissues were removed at the time of transplantation or death from non‐IPF and IPF patients. The tissue samples were stripped of all identifiers and designated as waste (exemption 4). Written informed consent was obtained on all patients or their surrogates prior to the procedure being performed. Use of human lung tissues was approved by the Institutional Review Board (IRB) at the Ohio State University (protocols 2017H0309 and 2021H0180) and all identifiers other than the diagnosis  were removed from the specimens provided. Normal fibroblasts were isolated from the normal lungs of decedents with no evidence of pulmonary disease who were HIV, hepatitis C, and SARS‐CoV‐2 negative and had no evidence of significant trauma. For the preparation of IPF and control lung fibroblasts, the visceral pleura was dissected and removed. The parenchyma was washed with PBS to eliminate RBCs. Minced tissue was transferred to GentleMACs C‐Tubes containing the enzymatic cocktail  and incubated at 37°C. After 1 h, cold serum was added to reduce the enzymatic activity. The filtered lung cell suspension was resuspended in RBC lysis solution, incubated for 7 min, and plated in a 75 cm flask for 24 h. Nonadherent cells were removed, and fresh media was added every 2 days. Experiments were conducted with isolated control and IPF fibroblasts between passages 4 and 7.2.25 control and IPF fibroblasts cultured in the normoxic or hypoxic chambers for 4 days were treated with the GPR‐81 antagonist 3 hydroxy butyric acid  or TGF‐β (2 ng/mL) for an additional 24 h. For silencing, control and IPF fibroblasts were transfected with 50 nM FoxM1, LDHA, or GPR‐81 siRNA  using Lipofectamine 3000 . Scrambled siRNA was used as a negative control . Control and IPF fibroblasts (3 × 105 cells/30 mm cell culture dish) were lysed with 1 × cell lysis buffer  containing protease inhibitor  and phosphatase inhibitor , and western blotting was performed. Cell lysates were collected, sonicated on ice for 15 s, and then denatured using 3× Laemmli buffer at 95°C for 3 min. Protein quantification was conducted by DC™ Protein Assay Kit I . Protein samples (5 to 15 μg) were loaded and separated through the gradient polyacrylamide gels (Novex WedgeWell Tris‐Glycine Gel) having 4%–20% polyacrylamide concentration . Precision Plus Protein Kaleidoscope Prestained Protein Standards  were used as a protein marker. Proteins on the gel were then electrically transferred to a PVDF membrane  using a Protean III tank transfer system (Bio‐Rad). After blocking with 5% bovine serum albumin in 1× TBST  for 1 h at room temperature, the membrane was incubated with antibodies  as follows: FoxM1  for antigen retrieval. All slides were then transferred to a humid staining rack in distilled water. All tissue on slides were circled with an immunopen, and then two drops of TBS were added to each slide and left to sit for 5 min. Endogenous peroxidases were quenched with 3% hydrogen peroxide in PBS for 5 min and sections were incubated with diluted normal swine serum to block nonspecific binding of secondary antibodies for 15 min at room temperature. The slides were incubated overnight at 4°C with 1:200 dilution of LDHB  or 1:100 dilution of GPR‐81 . After incubation, the slides were washed with PBS and then incubated with goat anti‐rabbit biotin‐conjugated secondary antibody  for 60 min (1:1500 dilution). After washing with TBS, the slides were incubated in HRP‐Streptavidin solution (Vector Laboratories) for 45 min and subjected to the application of the chromogen substrate 3, 3′ Diaminobenzene for 3 min and counterstained with hematoxylin. Slides were then put into a water bath with running tap water and then were rinsed in PBS before being dehydrated through a graded series of ethanol. Slides were then put into xylene before being treated with permanent mounting medium and coverslips being applied. Slides were then left to dry overnight. Images were obtained using a EVOS M7000 microscope (Invitrogen) and processed with Celleste Image analysis version 5.0 software. Control specimens were processed under the same conditions without the primary antibody to produce negative control (NC) sections.2.32, 5% CO2, and 94% N2. Control and IPF fibroblasts (2 × 104 cells/well of a 96‐well plate) were cultured in serum containing DMEM medium under the normoxic and hypoxic conditions for 5 days. To minimize the potential for reoxygenation, the cells were cultured in the hypoxic conditions without interruption. To precisely measure extracellular lactate levels, control and IPF fibroblasts were incubated in the hypoxic chamber for 5 days without changing the medium. For the measurement of cell viability, control and IPF fibroblasts cultured under normoxic and hypoxic conditions as indicated were incubated with 20 μL of Cell Titer Blue reagent  for 3 h, and cell viability was measured at 560 nm (Ex)/590 nm (Em) of fluorescence using a 96‐well plate reader . Extracellular lactate levels were measured using a Lactate Assay kit . Briefly, extracellular medium derived from control and IPF fibroblasts cultured under the conditions as described above was collected. Lactate standard was prepared and the final concentration of lactate (ng/μl) was measured according to the manufacturer's protocol.Control and IPF fibroblasts were cultured at 37°C in a modular hypoxic chamber  flushed with a gas mixture containing 1% O2.4t test (two‐tailed), and two sample equal variance was used. Significance level was set at p < 0.05.Data are expressed as the means ± SD. Two‐dimensional column graphs were prepared using Microsoft Excel. Protein expression levels are also presented as a box‐whisker plot showing the lowest expression, lower quartile, median, upper quartile, and the highest expression using SPSS v.19. N equals the number of unique and independent experimental samples. The significance of the sample data was determined using student 33.1Prior studies have linked hypoxia to the pathogenesis of fibrotic diseases including lung fibrosis  regulate the interconversion between pyruvate and lactate  have been implicated in the pathogenesis of pulmonary fibrosis  is known to be activated leading to increased production of lactate despite an ample supply of oxygen Devic, . In lungIPF is a disease of aging and it has been demonstrated that IPF fibroblasts exhibit a senescent phenotype. However, it is not clear how senescent IPF fibroblasts contribute to the maintenance and propagation of the persistent wound‐repair response that leads to progressive lung fibrosis. It has been postulated that senescent fibroblasts influence the phenotype of other cells via paracrine mechanisms in fibrosis and cancer; these effects have been attributed to the senescence‐associated secretory phenotype  leading to phosphorylation of the common co‐SMAD, SMAD4, which traffics to the nucleus and serves as a transcription factor. Transcriptional responses to SMAD4 can be modulated by co‐activators and corepressors, including serum response factor (SRF) and myocardin‐related transcription factor‐A (MRTF‐A), both of which have been implicated in myofibroblast differentiation. We have shown that SMAD‐mediated activation of the non‐receptor tyrosine kinase, focal adhesion kinase (FAK), is essential for TGF‐β‐induced myofibroblast activation, that upregulation of α‐SMA is evident within 12 h of treatment, and that formation of organized alpha smooth muscle actin stress fibers, indicative of the myofibroblast phenotype, occurs within 24 h of treatment under the culture conditions used in this study , although the extent of α‐SMA induction was reduced compared to that from non‐pH adjusted medium. Thus, our findings suggest that lactate signaling through GPR‐81 functions as an additional mechanism, along with the potential effects of TGF‐β activation and direct regulation of pH sensors in the induction of myofibroblast differentiation. As lung tissues of patients with IPF have enhanced lactic acid concentrations, we suggest that each of these mechanisms may have a role in propagation of the myofibroblast phenotype and contribute to progressive pulmonary fibrosis in vivo.Intercellular communication and cellular reprogramming are key underlying mechanisms of lung fibrogenesis. Such paracrine signaling for the induction of myofibroblasts may account for the geographically distinct development of fibroblast foci described in IPF , NIH/NHLBI HL141195 (JCH), and 1 U01 HL14555‐01(MR).All authors declare no conflict of interest.This study was approved by the Ethics Committee of the Ohio State University. The use of human lung tissues was approved by the Institutional Review Board (IRB) at the Ohio State University (protocols 2017H0309 and 2021H0180) and all identifiers other than the diagnosis  were removed from the specimens provided.https://figshare.com/s/8b30179f7f1b085292efURL: https://doi.org/10.6084/m9.figshare.22189201DOI: Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Figure S3.Click here for additional data file.Figure S4.Click here for additional data file.Figure S5.Click here for additional data file.Figure S6.Click here for additional data file.


GRN). Modeling TDP-43 pathology has been challenging in vivo and in vitro. We present a three-dimensional induced pluripotent stem cell (iPSC)-derived paradigm—mature brain organoids (mbOrg)—composed of cortical-like-astrocytes (iA) and neurons. When devoid of GRN, mbOrgs spontaneously recapitulate TDP-43 mis-localization, hyperphosphorylation, and LoF phenotypes. Mixing and matching genotypes in mbOrgs showed that GRN−/− iA are drivers for TDP-43 pathology. Finally, we rescued TDP-43 LoF by adding exogenous progranulin, demonstrating a link between TDP-43 LoF and progranulin expression. In conclusion, we present an iPSC-derived platform that shows striking features of human TDP-43 proteinopathy and provides a tool for the mechanistic modeling of TDP-43 pathology and patient-tailored therapeutic screening for FTD and ALS.Loss of function (LoF) of TAR-DNA binding protein 43 (TDP-43) and mis-localization, together with TDP-43-positive and hyperphosphorylated inclusions, are found in post-mortem tissue of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those carrying LoF variants in the progranulin gene (  •GRN−/− mbOrgs made of mature astrocytes and neurons show traits of TDP-43 pathology•GRN−/− astrocytes are necessary and sufficient for STMN2 mis-splicing in mbOrg•STMN2 mis-splicing is rescued, adding progranulin to neuron and astrocyte co-cultures GRN expression, these cultures present features of ALS- and FTD-associated pathology hardly ever observed in vitro. These phenotypes are shown to be primarily driven by diseased astrocytes and can be rescued by pro-granulin supplementation.In this article, de Majo et al. present a novel 3D iPSC-derived model to study neurodegenerative disorders such as ALS and FTD. When devoid of  TARDBP) gene encodes for the TDP-43 protein, which plays a pivotal role in these two devastating neurodegenerative disorders. TDP-43 was also recently implicated in limbic-predominant age-related TDP-43 encephalopathy (LATE) (STMN2), a neuron-specific gene important for neuronal survival  and amyotrophic lateral sclerosis (ALS) are two fatal neurodegenerative diseases. ALS is estimated to affect 2.1 cases per 100,000 people per year , while Fy (LATE) . Althougy (LATE) . Loss ofsurvival .GRN) gene. (GRN transcripts are composed of 13 exons and encode for the progranulin (PGRN) protein, a highly conserved, approximately 80-kDa protein involved in lysosomal function, neuronal survival, and inflammation  gene. . GRN traammation . The vasmination . The GRNdiseases in vitro or in vivo has been a challenge, with most in vitro models applying exogenous stress to replicate pathology akin to what is observed in post-mortem ALS/FTD central nervous system (CNS) tissue. Within these paradigms, induced pluripotent stem cells (iPSCs) hold great promise; they allow for patient- and tissue-specific human-derived in vitro models, which can be used for therapeutic screening and phenotype testing (GRN loss of function (LoF)  . GRN depignaling  and cellignaling . Interespatients . One poss manner ; 2016. Rs manner . Indeed,neration . The rolGRN LoF in FTD. When devoid of granulin expression (GRN−/−), our model develops features of TDP-43 pathology, including cryptic STMN2 (CrSTMN2) splicing, and extranuclear and hyperphosphorylated TDP-43 inclusions. This study presents the first in vitro model showing robust evidence of numerous FTD/ALS pathology markers spontaneously developing, overcoming the need of exogeneous chemical-induced stress or overexpression. Furthermore, we obtained partial phenotype rescue when GRN−/− cells were treated with exogenous full-length PGRN, demonstrating that the development of TDP-43 pathology depends on PGRN expression. We believe this model could provide insight into cell biological mechanisms, leading to TDP-43 pathology and offer a platform for patient-tailored phenotype and therapeutic screening for FTD and ALS patients with suspected or confirmed TDP-43 proteinopathy.To address this challenge, we applied our previously developed iPSC-derived 3D co-culture model  composedGRN LoF, we modified a previously described protocol to study glial-neuronal interactions (GRN+/+) iPSCs or isogenic GRN knockout (GRN−/−) iPSCs , an astrocyte specific gene well expressed by astrocytes  iPSCs G. Import of PGRN B and S1E neurons B and 1C.trocytes , was str− mbOrgs A. Simila− mbOrgs B. Both rGRN-associated FTD TDP-43 proteinopathy is characterized by increased levels of phospho-TDP-43 (pTDP-43) and abnormal TDP-43 cytoplasmic accumulation, seen in postmortem CNS tissue  has been considered a robust indicator of TDP-43 pathology and correlates with the level of pTDP-43 . Immunostaining for TDP-43 showed expected pathology in the both-cell-type GRN−/− mbOrgs. We also found TDP-43 pathology in the GRN+/+ neurons + GRN−/− astrocytes mbOrgs  cultures. The results point to a profound deficit in synaptosome phagocytosis in the GRN−/− astrocytes  and post-synaptic marker post-synaptic density protein 95 (PSD-95). Confocal microscopy analysis and IMARIS 3D reconstruction showed that in comparison with 4-week-old GRN+/+ mbOrgs. GRN−/− mbOrgs contain more SYP and PSD-95 puncta   B. When i(28 DIV) B. As pre neurons G. This rl bodies F. We nex neurons G. These in vitro human iPSC-derived neuro-glia 3D model of TDP-43 proteinopathy in a context of PGRN deficiency. While iPSC-induced neurons have provided numerous cell-autonomous biological insights into neurodegeneration, most models do not recapitulate multiple aspects of TDP-43-associated pathology. These phenotypic challenges have made therapeutic discovery difficult for TDP-43 proteinopathies Further resources and reagents inquiries should be directed to and will be fulfilled by the corresponding authors . If normally distributed, two-tailed unpaired Student’s t test was performed. If normally distributed, a one-way ANOVA was performed. A p value of less than 0.05 was considered significant. If non-normally distributed, a Mann-Whitney U-test was performed. A p value of less than 0.05 was considered significant. Using DESeq2, Wald tests were performed to evaluate genes for differential expression between conditions. The false discovery rate multiple testing correction method was then used for adjusted p values.−/− iPSC lines. M.dM, E.M.U., E.M., H.L., and K.S. conceived the hypothesis. E.M., J.R.K, M.Kurnellas, M.Kampmann, M.E.W., E.J.H., and E.M.U. provided resources. M.dM., M.Koontz, E.M., and E.M.U. wrote the manuscript. All authors reviewed and approved the manuscript.M.dM., M.Koontz, and E.M.U. designed experiments. M.dM., M.Koontz, E.M., N.S., A.R., Y.K., S.L.G., N.M.D., and K.L. performed experiments and/or analyzed data. Y.M. generated the GRN


G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterises the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1 Amyotrophic Lateral Sclerosis (ALS) is characterised by a progressive loss of motor control due to a loss of motor neurons (MNs) in the spinal cord, brain stem, and motor cortex. Prior to MN loss, structural and functional changes in synapses have been reported between neurons in both the brain and spinal cord . SynaptoTAR-DNA-Binding Protein 43 (TDP-43) is a ubiquitously expressed DNA/RNA binding protein that provides critical roles in RNA splicing, translation and transport in the nucleus and cytoplasm of neurons and glia in the nervous system . MutatioTDP-43 may play a role at synapses in both healthy and diseased conditions . TDP-43-Taken together, synaptic dysfunction may be linked to TDP-43 pathology. However, there has been little characterisation of the synaptic expression of TDP-43 in spinal cord synapses. MNs, for example, receive numerous different types of synaptic inputs, from excitatory, inhibitory and modulatory synapses derived from descending inputs from the brain, local spinal cord interneurons and sensory neurons. Not all synapses are equal, as some may be more selectively targeted in certain disease conditions . UnderstIn this study, we have used high-resolution and super-resolution microscopy techniques to visualise and assess the presence of phosphorylated TDP-43 (pTDP-43) at synapses within the mouse spinal cord.All procedures performed on animals were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 and were approved by the University of St Andrews Animal Welfare and Ethics Committee. The B6SJL-TgN(SOD1-G93A)1Gur/J (SOD1) mouse line was kindly provided by Dr. Richard Mead. PSD95-eGFP mice were originally obtained from Prof. Seth Grant (University of Edinburgh). The PSD95-eGFP mouse is a genetically engineered in-frame fusion knock-in mouse, bred on a C57BL/6J background, using a strategy detailed previously . The enhMice were anaesthetised with pentobarbitol , and the chest cavity opened to reveal the heart. The right atrium was severed and 10 mL ice cold 1 × phosphate buffered saline (PBS) was perfused through the left ventricle, followed by 10 mL 4% paraformaldehyde . The lumbar spinal cord was then dissected and incubated for a further 3–4 h (h) in 4% PFA before being incubated in sucrose 30% w/v for up to 72 h at 4°C until sunk. Prior to cryo-embedding, the spinal cord was cut at approximately Lumbar segment 1 (L1), which was determined as being approximately 2 mm rostral of the lumbar enlargement, and by examining the distribution of the ventral roots. Thus, the first cryosections were taken at approximately Lumbar segment 1–2 (L1-2). Tissue was then cryo-embedded in OCT compound and stored at − 80°C. Cryosections at 20 μm thickness were obtained using a Leica CM1860 cryostat and adhered to Superfrost Gold Plus glass slides (VWR). For experiments requiring total internal reflection fluorescence (TIRF) microscopy, 10 μm thick sections were adhered to an Argon Plasma cleaned coverslip (1.0 thickness).™ Glass Antifade Mountant (Invitrogen).For immunohistochemistry, slides with spinal cord slices were first heated at 37°C for 30 min to aid the adherence of the tissue to the glass slides and reduce tissue loss during subsequent wash steps. Slides were washed three times in PBS before being blocked and permeabilised in PBS containing 3% Bovine Serum Albumin (BSA), 1% donkey serum and 0.2% Triton X100 for 2 h at room temperature. Primary antibodies were diluted 1:500 in PBS containing 1.5% BSA and 0.1% Triton X100, and samples were incubated with primary antibody solution for 2 nights at 4°C. Primary antibodies used include: Anti-SMI32  Anti-VGLUT2 , anti-VGLUT1 , and two different anti-pTDP-43 antibodies . Once incubated, slides were washed five times in PBS over the course of 1–2 h. Secondary antibodies were diluted 1 in 500 in PBS with 0.1% Triton X100, and tissue was incubated in secondary antibody solution for 1.5–2 h at room temperature followed by a further five washes in PBS over the course of 1–2 h. Secondary antibodies used were Donkey-anti-Rabbit conjugated to Alexa Fluor 555 , Donkey-anti-Mouse conjugated to Alexa Fluor 647 , Donkey anti-Mouse conjugated to Alexa Fluor 405 plus  and Donkey anti-Guinea Pig conjugated to Alexa Fluor 647 . Finally, slides were dried and coverslips of 0.17 mm thickness were adhered using hard-setting Prolongin silico approach that performs large-scale predictions of protein-RNA interactions based on physico-chemical properties; the algorithm is called catRAPID  microscopy was performed using the Leica SP8 SMD g-STED microscope available at the Edinburgh Super-Resolution Imaging Consortium (ESRIC) hosted by Heriot Watt University. Excitation was provided by a CW super-continuum white light laser source. Depletion was provided by a 594 and 775 nm laser. Images were acquired with a 1.4 NA 100× oil STED objective lens. The optical zoom was set to provide a resultant image pixel size was 20 nm. Fluorescence was detected using a Leica Hybrid detector, gated at 0.5–8 ns for g-STED images. Confocal and g-STED images were captured sequentially  from regions of interest.−2). Fluorescence collected was separated from laser excitation using a dichroic mirror [Di01-R405/488/561/635 ], and was passed through appropriate filters . The fluorescence was then passed through a 2.5× beam expander and recorded on an EMCCD camera  operating in frame transfer mode (EMGain = 11.5 e−/ADU and 250 ADU/photon). The microscope was automated using the open-source microscopy platform Micromanager. TIRF images were acquired of PSD95-eGFP, VGLUT2 and VGLUT1 by acquiring 100 frames with a 50 ms exposure time. Super-resolution images of TDP-43 were generated using Aptamer DNA-PAINT (AD-PAINT)  was performed using an Inverted Nikon TI2 microscope  with a 1.49 NA 60x TIRF objective  and a ‘perfect focus’ system to autocorrect for z-drift during acquisition. Illumination was provided with 488 , 561  and 638 nm  laser lines  . Briefly2) of any of the four lines was too low, or minimum or maximum sizes of the clusters were out-with the expected ranges.Image analysis was conducted in Fiji Is Just ImageJ (FIJI) . As a stTo quantify the synaptic expression of pTDP-43 from the Airyscan data set, images underwent background subtraction, Gaussian blurring and manual thresholding to segment structures in each image. A small, customised macro was written to process each image see , 2022. AIn 3D data sets , images From the SMLM data sets, images were assessed for drift prior to analysis and excluded accordingly. TDP-43-aptamer images first underwent a background subtraction . Using the GDSC SMLM plugin, the Peak Fit function was used to detect single molecule fluorescence events to a Gaussian fit. Localisations were filtered based on a minimum signal strength threshold of 20, a minimum photon count threshold of 50, and a fitting precision threshold of 40 nm. Clusters of TDP-43 from single molecule localisation events were detected using Density-Based Cluster Analysis (DBSCAN) , and their association with synaptic structures was quantified from their spatial overlap with manually thresholded VGLUT1 and VGLUT2 terminals.T-Test was used to compare measures of parameters between two groups (U test was used (U Test (p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). Samples sizes are typically denoted in the figures, either as N = number of animals, or n = number of synapses or clusters.Data handling and preparation of graphs was performed in Microsoft Excel. Statistical tests were performed in GraphPad Prism (Version 9.1.0 (21)) or IBM SPSS (Version 28.0.1.1 (15)). Bar charts display error bars denoting the standard error of the mean. Violin plots are used with an inclusive median. Shapiro–Wilk test was used to assess data distribution for normality. A parametric Two-sample o groups –F, 4C–F;was used , 4K,M. Awas used . When anwas used –I. A Chiwas used , 3C. Com  obtained from adult (111 days old) control mice that were heterozygous for the PSD95-eGFP knock-in. Two different polyclonal antibodies, raised in rabbit, were tested for labelling pTDP-43 (serine 409/410) in the mouse spinal cord . Both antibodies displayed similar labelling patterns in the mouse spinal cord, with some apparent nuclear and somatic labelling of neuronal cells, as well a dense array of puncta in the neuropil that were not present in the negative primary antibody control labelling . The majUsing high-resolution confocal microscopy, we found that pTDP-43 displays strong labelling of large neurons in the ventral horn of the mouse spinal cord . pTDP-43n = 615 clusters, median: 297 nm; n = 947 clusters, median: 211 nm; n = 214 clusters, median: 140 nm; We next characterised the morphology of these pTDP-43 clusters in the neuropil, as assessed using a number of different high-resolution and super-resolution microscopy techniques. These included Zeiss Airyscan, Leica Confocal and g-STED microscopes . The diaU = 188965, p = 0.144), displaying a mean of 318 ± 64 nm  appeared very similar in Confocal microscopy compared to those detected using the Cosmo Bio antibody  of excitatory synapses ,B. PSD95N = 5 mice). pTDP-43 clusters were identified in 87.6 ± 3.7% of VGLUT1-positive presynaptic terminals, compared with 59.6 ± 5.0% of VGLUT2-positive presynaptic terminals [F = 19.9, p < 0.0001; T(8) = 15.2, p < 0.0001; T(8) = 7.1, p < 0.001; Using Airyscan microscopy, images of pTDP-43 clusters in excitatory synapses were captured from the ventral horn of lumbar spinal cord sections from healthy control mice (T(8) = 1.7, p = 0.121; When pTDP-43 cluster size was determined using thresholding to measure individual cluster area, pTDP-43 clusters were not statistically different in their sizes between VGLUT1 and VGLUT2-associated presynaptic terminals, although there was a non-significant trend toward increased cluster size in presynaptic VGLUT2 compartments [F = 26.1, p < 0.001; F = 19.2, p < 0.001; These findings were replicated in a complementary set of experiments. The pTDP-43 clusters were instead labelled using the ProteinTech antibody, VGLUT1 and VGLUT2 labelling was performed separately in different tissue sections, and the data was collected and analysed using 3D data sets acquired using the Airyscan . From thp < 0.001; U = 101474.5, p < 0.001; We also used confocal/g-STED microscopy to visualise the synaptic expression of pTDP-43 clusters in the ventral horn of one mouse spinal cord tissue section with greater resolution. Confocal images were obtained of VGLUT1, VGLUT2 and PSD95-eGFP to visualise excitatory presynaptic terminals and the ‘PSDs’ of excitatory synapses . g-STED These data confirm that pTDP-43 is localised synaptically and that there is heterogeneity in the presence of pTDP-43 clusters between different, distinct subtypes of synapses and across synaptic compartments.p = 0.004; Aptamers are sequences of DNA that can be specifically designed to bind to peptide sequences of interest. An anti-TDP-43 aptamer has been developed and tested for use in identifying TDP-43 aggregation . AD-PAINVarious reports indicate TDP-43 pathology may play a role in the synaptic dysfunction in ALS. The SOD1 ALS mouse model displays many cellular and behavioural phenotypes that recapitulate the human disease, including synaptic dysfunction, motor neuron loss and progressive motor control deficits. Although many reports indicate that the SOD1 mouse model displays no TDP-43-associated pathology, the synaptic presence of TDP-43 in this model has not been investigated using nanoscopy techniques that may detect more subtle changes.N = 5) and SOD1 mice (N = 6). The SOD1 mice were cross-bred with PSD95-eGFP mice such that all progeny were heterozygous for the PSD95-eGFP mutation, with some of the progeny expressing the SOD1 mutation. The control littermates not expressing the SOD1 mutation were the ones used in the previous sections  = 2.9, p = 0.017; T(9) = 2.4, p = 0.041; T(9) = 1.3, p = 0.238; T(9) = 1.7, p = 0.123; VGLUT1-associated presynaptic terminals were significantly smaller in the SOD1 spinal cord tissue compared to control [F = 1.0, p = 0.319; F = 1.0, p = 0.322, F = 0.2, p = 0.686, U = 52,480, p = 0.638; U = 176,092, p = 0.300; N = 5 SOD1 samples for the VGLUT1 labelling and N = 6 SOD1 samples for the VGLUT2 labelling, we also observed no significant differences in percentages of synapses with pTDP-43 [F = 3.0, p = 0.092; F = 1.0, p = 0.325; A Two-Way ANOVA was used to analyse differences in the association of pTDP-43 clusters with synapses between the two disease conditions (Control versus SOD1) and between different synapse structure types . The data were plotted as the percentage difference between SOD1 and Control mice . There wOur study reveals nanoscale synaptic expression of TDP-43 in the ventral horn of the lumbar spinal cord in adult mice. This was apparent using two different anti-pTDP-43 antibodies and an anti-TDP-43 aptamer, visualised with a range of high-resolution and super-resolution microscopy techniques. While there is considerable literature on the role of TDP-43 in synaptic function in health and disease , our finEach method incurs its own advantages and limitations. While AD-PAINT provides optimal resolution of TDP-43 clusters, image acquisition is slow and large data sets are generated that require considerable computational power (>1Gb per acquisition), making processing and analysis complex and time-consuming. Both the Airyscan and g-STED methods offered reasonable improvements in resolution beyond diffraction limited confocal microscopy (though inferior to AD-PAINT) and are relatively ‘user friendly’ for non-specialist microscopists in terms of sample preparation, speed of acquisition, data-set size and analysis.Nanoscale clusters of TDP-43 were found in both VGLUT1- and VGLUT2-associated presynaptic terminals in regions where motor neurons reside. While TDP-43 appeared to be expressed more predominantly in presynaptic terminals, our data did indicate the presence of postsynaptic TDP-43 clusters, as evidenced by its partial colocalization with PSD95, which has also been observed in other studies . The morDespite VGLUT1- synapses being more commonly associated with a greater number of pTDP-43 clusters, such clusters were slightly larger in VGLUT2-associated synapses, when measured using g-STED microscopy. Recent findings using super-resolution microscopy discerned that larger inclusions of TDP-43, not smaller ones, were more strongly associated with neuronal toxicity , furtherG93a mouse model. Our data do not reveal any significant difference in the cluster size or synaptic expression of pTDP-43 in SOD1 mice compared to healthy littermate controls. Previous reports have provided conflicting results regarding the presence or timescale of TDP-43 pathology in the SOD1Similar to previous observations by ourselves and others, we observed that the PSDs of excitatory synapses are reduced in size in the SOD1 mice. Interestingly this effect was most prominent in the VGLUT1-associated synapses – with a corresponding reduction in the sizes of the VGLUT1 presynaptic terminals. This finding is mirrored by the heterogeneity in TDP-43 between different synapses, whereby it is more frequently associated with VGLUT1 synapses compared to VGLUT2 synapses. Other synapse subtypes received by motor neurons are also subject to changes, including inhibitory glycinergic synapses  and moduMutations in a number of different genes, including C9ORF72, TDP43, FUS and SOD1, can all lead to some form of motor neuron disorder or the related cognitive disorder frontotemporal dementia. It is possible that the synapse may act as a key subcellular locus of convergence for these multiple different disease pathways. Recent proteomics studies from human ALS patients identified over 30 different ALS-associated proteins in biochemically extracted synaptosomes . For exaIn summary, our quantitative super-resolution imaging provides novel characterisation of the subcellular distribution of TDP-43 and nanoscale organisation of protein clusters. These techniques are valuable for understanding the role of TDP-43 in neuronal function. The sensitivity and resolution of such techniques will be valuable in identifying subtle early-stage aggregations of TDP-43, as it is known that complex aggregates form during early pathogenesis of ALS . Such inThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the University of St Andrews Animal Welfare and Ethics Committee.MB conceived the hypothesis, wrote the grants to support the research, performed and directed IHC experiments, imaging experiments and analysis, and wrote the manuscript. AA-H, KD and LM performed IHC experiments and high-resolution microscopy and analysis. OK performed IHC experiments, TDP-43 aptamer experiments, and SMLM experiments FZ created PSD-95-eGFP mouse model. SG created PSD-95-eGFP mouse model and feedback on manuscript. MH wrote grants to support the research, performed TDP-43 aptamer experiments and SMLM experiments, assisted with analysis and manuscript writing. GM wrote grants to support the research and assisted with manuscript writing. All authors contributed to the article and approved the submitted version.This work was supported by Motor Neurone Disease (MND) Association UK (Miles/Apr18/863-791), Chief Scientist Office, RS Macdonald Charitable Trust, ALS CURE Project, the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (695568 SYNNOVATE), Simons Foundation Autism Research Initiative (529085), and the Wellcome Trust (Technology Development grant 202932).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


IV(L1–4)2] (1–4), featuringtridentate bi-negative ONS chelating S-alkyl/aryl-substituteddithiocarbazate ligands H2L1–4, are reported.All the synthesized non-oxido VIV compounds are characterizedby elemental analysis, spectroscopy ,ESI-MS, as well as electrochemical techniques (cyclic voltammetry).Single-crystal X-ray diffraction studies of 1–3 reveal that the mononuclear non-oxido VIV complexesshow distorted octahedral (1 and 2) or trigonalprismatic (3) arrangement around the non-oxido VIV center. EPR and DFT data indicate the coexistence of mer and fac isomers in solution, and ESI-MSresults suggest a partial oxidation of [VIV(L1–4)2] to [VV(L1–4)2]+ and [VVO2(L1–4)]−; therefore, all these three complexes are plausibleactive species. Complexes 1–4 interactwith bovine serum albumin (BSA) with a moderate binding affinity,and docking calculations reveal non-covalent interactions with differentregions of BSA, particularly with Tyr, Lys, Arg, and Thr residues. In vitro cytotoxic activity of all complexes is assayedagainst the HT-29 (colon cancer) and HeLa  cellsand compared with the NIH-3T3 (mouse embryonic fibroblast) normalcell line by MTT assay and DAPI staining. The results suggest thatcomplexes 1–4 are cytotoxic in natureand induce cell death in the cancer cell lines by apoptosis and thata mixture of VIV, VV, and VVO2 species could be responsible for the biological activity.A seriesof mononuclear non-oxido vanadium(IV) complexes, [V IV(L1−4)2] complexes oftridentate ONS dithiocarbazate ligands is reported. The results indicatethat a mixture of [VIV(L1−4)2], [VV(L1−4)2]+, and [VVO2(L1−4)2]− species is formed, which may be responsiblefor the observed activity.The synthesis, stability, and chemicaltransformations insolution, protein interaction, and cytotoxicity of a series of non-oxido[V Thereis interest in the study of the pharmaceutical roles of several vanadiumcomplexes,46 such as [bis(cyclopentadienyl)vanadium(IV)] dichloride, [VIVO(phen)(H2O)2]2+,47 and [VIVO(phen)2(SO4)]48 , which exhibitedpotent antitumor activity against the human nasopharyngeal carcinoma-KBcell line and the clonogenic NALM-6 cell line, respectively. However,more recently, the relevance of addressing the consequences of theprobable hydrolysis, ligand exchange, and redox reactions of complexesof labile metal ions in in vitro and in vivo conditions have been highlighted,52 emphasizing the need to properly disclose which are the relevantbiological active species. Namely, incubation media used in studieswith mammalian cells also contain appreciable amounts of bovine serumalbumin (BSA), which competes for the binding to the metal ions present,often changing the nature of metal-containing species that enter insidecells.52Some oxidovanadium(IV)complexes, particularly  (1–4), formed by S-alkyl/aryl substituted dithiocarbazateligands (H2L1–4). All these compoundswere characterized through elemental analysis, mass spectrometry,spectroscopic techniques, and cyclic voltammetry. Structural evidencefor the formation of non-oxido VIV complexes was obtainedby the SC-XRD characterization of [VIV(L1–3)2] (1–3). To the bestof our knowledge, this is the second study involving the structuralcharacterization by single-crystal X-ray diffraction analysis (SC-XRD)of non-oxido vanadium(IV) complexes with ONS donor ligands.53Considering the potential therapeutic use of this type of compoundsand our expertise in the field of biological and pharmacological applicationsof vanadium compounds,and so forth, which have limited the experimentation ofV-based drugs in clinical practice.65 For example, recently, focushas been given to the products formed under biological media by thehydrolysis/oxidation of non-oxido VIV-complexes, whichhave demonstrated to be responsible for the biological properties.66 Thus, the possibility of hydrolysis and redox processes of new non-oxidoand oxidovanadium(IV) complexes is worth of being explored. Therefore,in this work, we address the chemical transformations, disclosingthe solution phase stability of complexes [VIV(L1–4)2] through various physico-chemical techniques. The synthesizedcomplexes 1–4 were also tested fortheir interaction with BSA and in vitro antiproliferativeactivity on HT-29 (colon cancer), HeLa , and NIH-3T3(mouse embryonic fibroblast) cell lines.Under biological conditions, vanadiumcomplexes often undergo chemicaltransformations like hydrolysis, redox reactions, ligand-exchange, IVO(acac)2], where acac stands for acetylacetonate, was prepared asdescribed in the literature.99 Reagentgrade solvents were dried and distilled under nitrogen prior to useaccording to standard procedures.100 HPLCgrade DMSO and CH3CN were used for spectroscopic and electrochemicalstudies, whereas EtOH and CH3CN were employed for synthesisof ligand precursors101 and metal complexes,respectively. BSA for the study of interactions with the compoundswas purchased from Sigma-Aldrich. Cell lines were obtained from National Centre of Cell Science (NCCS), Pune, India.Dulbecco’s phosphate buffered saline (DPBS), Dulbecco’smodified eagle medium (DMEM), fetal bovine serum (FBS), antibiotic-antimitoticsolution, and trypsin EDTA solution were procured from Himedia, Mumbai,India, while 4′,6-diamidino-2-phenylindole dihydrochloride(DAPI) and 3--2,5-diphenyltetrazolium (MTT)were purchased from Sigma-Aldrich, India. Elemental analysis measurementswere performed on A Vario EL cube CHNS elemental analyzer. A PerkinElmerSpectrum RXI spectrophotometer was used for recording the IR spectra,and 1H and 13C NMR spectra were recorded ona Bruker Ultrashield 400 MHz spectrometer in the presence of internalstandard SiMe4. Electronic spectra were obtained on a Lambda25PerkinElmer spectrophotometer. EPR spectra were recorded at 120 Kwith an X-band Bruker EMX spectrometer equipped with a HP 53150A microwavefrequency counter and a variable temperature unit, the instrumentalsetting being: microwave frequency 9.40–9.41 GHz; microwavepower 20 mW; time constant 81.92 ms; modulation frequency 100 kHz;modulation amplitude 4 Gauss. Positive- (+) and negative-ion (−)mode ESI-MS spectra were recorded with a high-resolution Q ExactivePlus Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific)with a flow rate of infusion into the ESI chamber of 5.00 μL/minand a recording range of 50–1000 m/z. The instrumental conditions for positive-ion mode spectrawere: spray voltage 2300 V; capillary temperature 250 °C; sheathgas 5–10 (arbitrary units); auxiliary gas 3 (arbitrary units);sweep gas 0 (arbitrary units); probe heater temperature 50 °C.For negative-ion mode spectra, they were: spray voltage −1900V; capillary temperature 250 °C; sheath gas 20 (arbitrary units);auxiliary gas 5 (arbitrary units); sweep gas 0 (arbitrary units);probe heater temperature 14 °C. The redox properties were studiedunder nitrogen by cyclic voltammetry using a three-compartment cellequipped with Pt wire (working and auxiliary) and Ag wire (reference)electrodes, interfaced with VoltaLab PST050 equipment. The cyclicvoltammograms of complexes 1–4 andfree ligands (H2L3–4) were obtained fromelectrolyte solutions of NBu4BF4 in CH2Cl2 (0.10 M). The potentials were measured in Volt (±10mV) vs saturated calomel electrode (SCE) at 200 mV/sscan rate using [Fe(η5-C5H5)2]0/+ as an internal reference.Most chemicals were obtainedfrom commercial sources. Carbon disulfide (HiMedia), hydrazine hydrate(Loba Chemie), methyl iodide (Sigma-Aldrich), benzyl chloride, and4-(diethylamino)salicylaldehyde (Sigma-Aldrich) and 2-hydroxy-1-naphthaldehyde(Sigma-Aldrich) were used as received. [V2L1–2 wereprepared in a fair yield by the condensation of 4-(diethylamino)salicylaldehydewith the corresponding S-alkyl/aryl substituted dithiocarbazates [S-methyldithiocarbazate(H2L1) and S-benzyldithiocarbazate (H2L2)], while H2L3–4 were synthesizedby the reaction of 2-hydroxy-1-naphthaldehyde with the respectivedithiocarbazates [S-methyldithiocarbazate (H2L3) and S-benzyldithiocarbazate (H2L4)] in a1:1 ratio in dry ethanol by adopting a reported procedure.102 The resulting yellowish compounds were filteredand then washed with ethanol and finally dried over fused CaCl2 in a desiccator.Thedithiocarbazate compounds H13H19N3OS2: C, 52.49;H, 6.44; N, 14.13; S, 21.56. Found: C, 52.28; H, 6.72; N, 14.48; S,21.23. IR : 3642 ν(O–H);3189 ν(N–H); 1630 ν(C=N); 1494 ν(C–O/phenolate);1317 ν(C=S). 1H NMR : δ (ppm) 13.13 , 10.13 , 8.31 , 7.31–6.12 , 3.31 , 2.51 , 1.11 . 13C NMR : δ (ppm)194.48, 159.79, 151.31, 147.81, 131.13, 106.51, 104.86, 97.66, 44.35,17.12, 12.98.Yield:74%. Anal. Calcdfor C19H23N3OS2: C, 61.09;H, 6.21; N, 11.25; S, 17.17. Found: C, 61.28; H, 6.28; N, 11.41; S,17.49. IR : 3683 ν(O–H);3109 ν(N–H); 1624 ν(C=N); 1516 ν(C–O/phenolate);1340 ν(C=S). 1H NMR : δ (ppm) 13.18 , 10.09 , 8.31 , 7.42–6.07 , 4.49 , 3.36 , 1.08 . 13C NMR: δ (ppm) 192.70,159.76, 151.36, 147.74, 137.45, 130.89, 129.64, 129.53, 129.45, 106.46,104.87, 97.54, 44.35, 37.85, 12.95.Yield:82%. Anal. CalcdC13H12N2OS2: C, 56.49;H, 4.38; N, 10.14; S, 23.20. Found: C, 56.53; H, 4.40; N, 10.20 S,23.18. IR : 3445 ν(O–H);3156 ν(N–H); 1620 ν(C=N); 1577 ν(C–O/phenolate);1325 ν(C=S). 1H NMR : δ (ppm) 13.42 , 11.08 , 9.19 , 8.80–7.23 , 2.50 . 13C NMR: δ (ppm) 196.72,158.69, 146.23, 134.21, 131.72, 129.36, 128.71, 128.61, 124.18, 123.73,118.70, 109.83, 17.38.Yield: 76%. Anal. CalcdC19H16N2OS2: C, 64.74;H, 4.58; N, 7.95; S, 18.19. Found: C, 64.80; H, 4.55; N, 7.96; S,18.23. IR : 3458 ν(O–H);3157 ν(N–H); 1622 ν(C=N); 1572 ν(C–O/phenolate);1327 ν(C=S). 1H NMR : δ (ppm) 13.46 , 11.09 , 9.20 , 8.71–7.21 , 4.59 . 13C NMR: δ (ppm) 194.73,157.25, 145.20, 136.09, 132.66, 131.72, 130.55, 127.34, 127.20, 126.80,125.74, 124.60, 123.69, 120.19, 120.71, 117.25, 116.11, 112.89, 45.15.Yield:80%. Anal. CalcdCIV(L1–4)2],were synthesized by mixing the ONS donor dithiocarbazates H2L1–4 with [VIVO(acac)2] instoichiometric amounts (2:1 molar ratio) in CH3CN and allowingit to reflux for 3 h. Greenish black-colored crystalline compounds(1–4) were directly obtained from the reactionmixtures. They were then collected by filtration, washed properlywith hexane, and dried over CaCl2. Suitable crystals ofmonomeric 1–3 compounds for single-crystal X-raymeasurements were collected from their respective reaction pots containingCH3CN solvent.The non-oxido vanadium(IV) compounds,formulated as : 629 (472), 529 (659), 390 (2157). ESI-MS (CH3CN): m/z 664.0960 [M + Na]+.Yield: 70%. Anal. Calcd for C38H42N6O2S4V: C, 57.48;H, 5.33; N, 10.58; S, 16.15.Found: C, 57.51; H, 5.34; N, 10.63; S, 16.13. IR : 1613 ν(C=N). UV–vis (DMSO): 628 (450), 526 (730), 401 (2046). ESI-MS (CH3CN): m/z 816.1585 [M + Na]+.Yield: 76%. Anal. Calcd for C26H20N4O2S4V: C, 52.08;H, 3.36; N, 9.34; S, 21.39.Found: C, 52.09; H, 3.33; N, 9.29; S, 21.40. IR : 1592 ν(C=N). UV–vis (DMSO): 653 (397), 522 (472), 382 (1869), 333 (2487).ESI-MS (CH3CN): m/z 621.9814[M + Na]+.Yield: 72%. Anal. Calcd for C38H28N4O2S4V: C, 60.70;H, 3.75; N, 7.45; S, 17.06.Found: C, 60.73; H, 3.71; N, 7.49; S, 17.09. IR : 1602 ν(C=N). UV–vis (DMSO): 644 (401), 528 (611), 385 (1932), 334 (2531).ESI-MS (CH3CN): m/z 774.0427[M + Na]+.Yield: 74%. Anal. Calcd for C1–3 were collected on astandard Bruker Kappa APEX II CCD-based 4-circle X-ray diffractometerusing graphite-monochromated Mo-Kα radiation. Datawere corrected for Lorentz and polarization effects; no extinctioncorrections were applied, but absorption correction was taken intoaccount on a semi-empirical basis using equivalent scans with Rint = Σ|Fo2 –Fo2(mean)|/Σ[Fo2] and Rσ = Σ[σ(Fo2)]/Σ[Fo2].Crystal andintensity data oncomplexes 103) and refined by full-matrix least squares techniques againstFo2 (SHELXL-97 and SHELXL-2014). Atomic scatteringfactors were taken from International Tables for Crystallography.104 The final agreement indices are R1 = Σ||Fo| –|Fc||/Σ|Fo| and wR2 = {Σ[w(Fo2 – Fc2)2]/Σ[(wFo2)2]}1/2, and weightingfunction used is w = 1/[σ2(Fo2) + (aP)2 + bP] with P = (maxFo2 + 2Fc2)/3. Goof = S = {Σ[w(Fo2 – Fc2)2]/(n–p)}1/2, where n is the numberof reflections and p is the total number of parametersrefined. Although the hydrogen atoms could be localized in differenceFourier syntheses, those of the organic moieties were refined in geometricallyoptimized positions riding on the corresponding carbon atoms, withC–H distances at T = 293 K/100 K of 0.96/0.98Å (−CH3), 0.97/0.99 Å (−CH2−), 0.93/0.95 Å (−CHsp2), andcommon isotropic thermal displacement parameters for chemical equivalentH-atoms. Figures were drawn using DIAMOND105 and Mercury.106 In the ball-and-stickmodels, all atoms are drawn as thermal displacement ellipsoids ofthe 50% level with exception of the hydrogen atoms, which are shownas spheres of arbitrary radii. Hydrogen bonds are drawn in red asdashed sticks. Crystallographic data as well as structure solutionand refinement details are summarized in Table S1. XP  wasused for structure representations.Thestructures were solved by direct methods  were prepared in DMSO and further diluted (1:10) immediatelybefore running the experiments. Aliquots of each complex (ca. 80 μM in DMSO) were added directly to a cuvettecontaining the BSA solution (ca. 1–2 μM)in PBS buffer to obtain complex/BSA molar ratios ranging from 0.1to 3.5. After addition of the complex, the solutions were allowedto stand for 5 min before measurements. Fluorescence emission spectrawere measured at room temperature with λex = 295nm between 310 and 500 nm. Fluorescence spectra containing the sameamount of complex but no BSA were recorded and subtracted from eachcorresponding emission spectrum of the mixtures. The DMSO amount inthe titrations was always lower than 5% (v/v). UV–vis spectrawere measured for each solution, and the absorbance at excitationand emission wavelengths were used to make corrections for inner filtereffects and reabsorption.109Fatty acid free BSA stock solution was prepared inPBS buffer , and the concentration was determined spectrophotometricallyconsidering the extinction coefficient 44,309 M109F0 and F are thefluorescence intensities of BSA in the absenceand presence of quencher, respectively, [Q] is thequencher concentration (the vanadium complex in our systems), and KSV is the Stern–Volmer quenching constant,obtained from the slope of the vs [Q] plot.The Stern–Volmerequation was used to fit the fluorescencequenching dataKBC) were estimatedthrough linearization of the quenching data, considering the equilibriumbetween free and bound quencher molecules when they bind independentlyto n equivalent sites of BSAThe binding constants (IV(L1–4)2], non-oxido vanadium(V)[VV(L1–4)2]+, anddioxidovanadium(V) [VVO2(L1–4)]− complexes was optimized with Gaussian 16, rev.B01,110 at the DFT theory level using thehybrid B3P86 functional combined with the split-valence 6-311g basisset. This method has been successfully applied and discussed in theliterature for the geometry prediction of vanadium species.112 For all the structures, minima were verified through frequency calculations.The 51V hyperfine coupling tensor A of all [VIV(L1–4)2] complexes was predicted with ORCA software version 4.0115 using the B2PLYP functional and 6-311g basis set, as suggestedpreviously.114 The percent deviation (PD)of the absolute calculated value  from the absolute experimental value (|Az|exptl) was obtained as 100 × [/|Az|exptl]. UV–visvertical excitations were simulated on the time-dependent densityfunctional theory (TD-DFT) framework. The simulations were carriedout on the previously optimized geometries using the range separatedCAM-B3LYP functional and the 6-31+g(d) basis set, according to themethod proposed in the literature.53 Thepredicted electronic spectra were generated using Gabedit software,115 and the molecular orbitals (MOs) involved inthe transitions were simulated performing a Mulliken population analysis(MPA) with Gaussian 16 at the same level of theory used for the TD-DFTcalculations and identified with the AOMix package (vers. 6.52).116The geometry ofnon-oxido vanadium(IV)/[VV(L1–4)2]+ and [VVO2(L1–4)]− can interact only through second-coordinationsphere interactions, were carried out. The blind exploration was performedon the rigid protein, building four evaluation spheres of 20 Åcontaining globally the whole protein. Genetic algorithm (GA) parametershave been set in 50 GA runs and a minimum of 100,000 operations. Theother parameters of GA were set to default. The scoring (Fitness of GoldScore) was determined through the recent validated versionsof GoldScore accounting for vanadium-complexes surface interactions.121 The best solutions (binding poses), were evaluatedtaking into account the mean (Fmean) andthe highest value (Fmax) of the scoringand population of the cluster containing the best pose.Dockingassays toward BSAwere carried out with GOLD 5.8 software,2 incubator (5% CO2). Thereafter, cells were harvested from the logarithmic phaseand seeded into a 96-well plate at a concentration of 8 × 104 cells per well. After overnight seeding, cells were treatedwith different concentrations  ofthe tested ligands and vanadium complexes (H2L1–4 and 1–4) for 48 h. The compounds’stock solutions were prepared in DMSO. DMEM was used to prepare thefinal working concentration of the compounds, so that the final amountof DMSO is less than 2%. The effect of the complexes on the viabilityof the cells was evaluated through MTT dye reduction assay by measuringthe optical density at 595 nm.93 DMSO alone,used to dissolve the complexes, was also employed for the controlgroup treatment.HT-29 (colon cancer),HeLa ,and NIH-3T3 (mouse embryonic fibroblasts) cells were maintained inDMEM supplemented with 10% FBS, penicillin–streptomycin solutionin a humidified (95% humidity) CO93 Briefly, the cells(HeLa and HT-29) were treated with the tested compounds 1–4 at a concentration of 10 μM and were kept for 24 h. Afterthis, fixation of the cells was done, followed by washing with PBS.Subsequently, the cells were stained with DAPI and kept in dark for10 min. Finally, the cells were washed twice with PBS and taken forimaging in a Leica TCS SP8 confocal laser scanning microscope with25× magnification.Morphologicalchanges of thenucleus and its structural integrity during cell death were analyzedby the DAPI staining method.1 and 2 in HT-29 cells was evaluatedusing Annexin V-FITC Apoptosis Detection Kit (Abgenex) by a flow cytometer.125 HT-29 cells (3 × 105 cells/well) were culturedin a 6-well plate for 24 h. After the cells were adhered, differentconcentrations (5 and 10 μM) of the tested complexes were treatedand incubated for further 48 h. Subsequently, the cells were collectedby trypsinization and then suspended in 1X binding buffer. Finally,the suspension was stained with 5 μL of Annexin V and PI atroom temperature and incubated for 15 min in the dark. Then, the sampleswere immediately analyzed under a BD Accuri C6 flow cytometer.The apoptotic populationinduced bycomplexes 1–4) were synthesized by the reactionof tridentatedibasic ONS donor dithiocarbazates H2L1–4 with [VIVO(acac)2] in a 2:1 molar ratio inCH3CN medium complexes (N medium 1. All co1–4 are summarized in the 2L1–4).A band in the region 3683–3445 cm–1 can beassigned to the stretching vibration of the aromatic O–H ofthe free ligands, which is absent in the corresponding metal complexesdue to the deprotonation and coordination. The spectra exhibit thecharacteristic bands of the dithiocarbazate ligands, which includea strong signature band at ∼1630–1620 cm–1 of the ν(C=N) vibration,126 together with a strong band in the region 1577–1494 cm–1 due to the C–O phenolic group.53 These bands shift toward lower frequency uponcoordination to the metal center. The absence of the strong characteristicν(V=O) stretching band in the region 900–1000cm–1 indicates the formation of a non-oxido vanadiumcenter in 1–4.127The spectral data of compounds 1 , 2 , and 3  werecharacterized by SC-XRD techniques. An overview of the solid-stateconformation of all three complexes is shown in 1 = Ci, 2 = C1, and 3 = C2), thus exhibiting different symmetry elements.The solid-state structuresof via the O-atom of their deprotonatedhydroxyl-OH, the imine N-atom, and the S-atom of the sulfide moieties.In each case, the coordination spheres around the V atoms are stronglydistorted. The geometries may be best described as distorted octahedralfor 1 and 2 with the O- and S-atoms in cis- and the N-atoms in trans-positions,while 3 exhibits a distorted trigonal prismatic geometrywith the O-atoms in cis-positions at one edge ofthe prism, while the S- and N-atoms are cis to eachother on the other edges of the prism, as shown in Figure S1.Themolecular structures of all three compounds contain discretemonomeric vanadium(IV) species coordinated by two tridentate, twofoldnegative ONS-donor Schiff base ligands. The Schiff bases coordinateto vanadium 1 and 148.9(1)°/149.5(1)°for 2] are typical for the octahedral vanadium coordinationwith all three donor atoms in a meridional arrangement[ideal value: 180°], while the value of 125.3(1)° for 3 corresponds quite well with a trigonal-prismatic vanadiumcoordination [ideal value of a regular trigonal prism: 125°].Additionally, bond lengths are also affected. While differences aresmall (Δ = 0.009 Å) for the vanadium–sulfur distances[d(V–S) range from 2.3434(7) Å for 2 to 2.3524(6)Å for 1], those for the vanadium–oxygen andvanadium–nitrogen distances are much greater . To a certain extent, this may be ascribed to thedifferent temperatures at which the crystal structures were determined,but this is not fully consistent with the behavior of 1 (T = 100 K) and 2 and 3 (T = 296 K). Therefore, other effects must be takeninto account. Notably, EPR analysis and DFT simulations indicate thatat least two isomers with comparable stability, close to the meridional and facial limit, coexist inthe solutions, from where the solid complexes are isolated (vide infra); thus, the isomeric selection experimentallyobserved could be ascribed to stabilizing crystal packing effects.Here, it is important to highlight that the characterized isomersare defined referring to the limit cases represented by meridional and facial arrangement, even though complexes 1–3 correspond to intermediate structures.As a result of these different coordinationgeometries, bond lengthsand angles in the coordination spheres of the vanadium atoms differsignificantly 1. Namely1–3. The first one concerns thedifferent conformations of the two crystallographic independent ligandsfound in the crystal structure of 2, indicated with m in Figure S2), not only in the domain of the sulfidemoiety with their low rotation barriers around the sulfur–carbonsingle bonds, but to a lesser extent also associated to the conjugateddouble bond system, which obviously may adopt different orientationsfor coordination.In this context, we highlight two features of the crystal structuresof 1 where the formation of dimeric centrosymmetric supramoleculesresultsfrom the π-interaction of their phenyl moieties , which are almost coplanar to eachother with six intermolecular carbon–carbon distances rangingfrom 3.358 to 3.411 Å. Moreover, similar intermolecular distances(3.257 Å) are found for the nitrogen and carbon atoms attachedto the phenyl groups.A second feature is found in the crystal structureof 1–4 were recorded in DMSO andshow similarities. As a representative example, the UV–visspectrum of 3 is shown in 1, 2, and 4 are reported in Figure S4. In the wavelength range 330–650 nm, three absorptions wereobserved for complexes 1 and 2, whereasfour bands are detected for 3 and 4. Aspointed out in the literature, for non-oxido vanadium(IV) complexes,the UV–vis spectrum is dominated by ligand-to-metal chargetransfer (LMCT) transitions, with large values of the molar absorptioncoefficient , from MOs centered on theligands to V-d based orbitals.128 TD-DFT calculationscarried out on 1–4 confirmed thesefindings and indicated that all the absorption bands are stronglymixed and correspond to a combination of various electronic transitions.Electronicabsorption spectraof –1 cm–1) and 530–532 nm (ε = 265–1080 M–1 cm–1) correspond to excitations from ligand-centeredMOs, as from highly delocalized π orbitals  to V-dz2 or V-dxz.65 The bands at 398 nm for 1 and 2  andbetween 332 and 384 nm for 3 and 4 (withmolar absorption coefficients of range of 6402–4707 M–1 cm–1) are countersigned by values of ε significantlyhigher than 4000 M–1 cm–1.65 They are LMCT absorptions mixed with ligand-to-ligandcharge transfers. The principal transitions involve MOs with charactersV-dz2, V-dxz, and V-dyz for vanadium and MOswith π character for the ligands.In the visible region, the absorptions in the range624–642nm , and DMSO/H2O 90/10 (v/v) in the positive-and negative-ion mode. The ESI-MS(+) and ESI-MS(−) spectraof 1 in CH3CN, recorded with a vanadium concentrationof 50 μM, are reported in m/z = 641.11, 642.11, 664.10,and 680.07. No peaks ascribable to the free ligand were detected,and this indicates the stability of the complex to hydrolysis evenat these low metal concentrations. The peak at m/z 641.11 is assigned to the oxidized species [VV(L1)2]+ , while that at m/z 642.11 can be explained with [VIV(L1)2] + H+ and/or to the isotopic pattern of[VV(L1)2]+ . The high-resolution instrumentused in this work allowed us to distinguish between the two options;even though the simulation seems to be identical up to two decimalfigures, it is significantly different when four decimal figures areconsidered. In particular, a peak at m/z 642.1094 is expected for [VV(L1)2]+ and at m/z 642.1138for [VIV(L1)2] + H+,which therefore contribute to the signals in the range m/z 641–645 in a negligible mode . To the best of our knowledge, thisis one of the first cases in which ESI-MS allows to discriminate unambiguouslytwo complexes in two different metal oxidation states. Instead, thesignals at m/z 664.11 and at m/z 680.07 can be assigned without anyambiguity to the adducts [VIV(L1)2] + Na+ and [VIV(L1)2] + K+ . The values of m/z for all the species detected by ESI-MS are listed in IV(L1)2] (1) can undergopartial oxidation to [VV(L1)2]+.ESI-MS spectra of the compounds m/z = 378.02 (VO2(L1)]−, formed upon theoxidation of [VIV(L1)2]. The simulationof the isotopic pattern is represented in Figure S9.The negative-ionspectrum shows few signals and a major peak at = 378.02 3b. The s2–4 iscomparable, and the observed species are listed in 2O 90/10 (v/v) are globally similar, but it shouldbe mentioned that the presence of water decreases the solubility ofthe complexes and favors the oxidation to VV, as shownin Figure S10.The behavior of complexes 129 (ii) the tridentate coordination of the ONS ligand to V is quitestrong, and the hydrolysis occurs at low extension, both in CH3CN and in MeOH/H2O 90/10 (v/v), even at the lowmetal concentrations used for recording the spectra; (iii) a non-oxidovanadium(V) species is formed in solution; (iv) the formation of non-oxidovanadium(V), a hard metal ion, is rather unusual and few exampleshave been reported in the literature up to now;131 and (v) under our experimental conditions, [VIV(L1)2] forms adducts only with Na+ andK+ ions but not with H+.A few aspects deserveto be emphasized: (i) the possibility thatthe observed oxidation process partially occurs in-source, duringthe recording of the spectra, cannot be excluded;2L1–4 were recordedin DMSO-d6. These spectra exhibit a resonancein the range δ =13.46–13.13 ppm attributable to −NH groups,132 one singlet peak of the phenolic −OHgroups in the region δ = 11.09–10.08 ppm due to intermolecularhydrogen bonding, and another sharp singlet at δ = 9.20–8.31ppm due to the azomethine −CH protons.138 The aromatic proton signals of the free ligands are clearly observedin the expected range between δ 8.80 and 6.07 ppm. For the aliphaticprotons, two multiplets are observed in the range δ = 3.36–3.31ppm for −N–CH2 and δ = 1.08–1.11ppm for the −CH3 protons of the diethylamino fragmentin the case of H2L1–2. Singlets for theCH2S– protons of H2L2 andH2L4 are observed around ∼4.45 ppm,which are absent in the spectra of H2L1 andH2L3.132 CompoundsH2L1 and H2L3 containone extra singlet in the aliphatic region near 2.51 ppm due to the−SCH3 protons.126Proton NMR spectra of H1–4 were obtained from Bu4NBF4/CH2Cl2 (0.10 M) electrolyte solutionsunder dinitrogen at a scan rate of 200 mV s–1. Thepotentials were measured in Volt (±10 mV) vs SCE using [Fe(η5-C5H5)2]0/+ (E1/2ox = 0.475 V) as an internalreference. The redox potential data are summarized and displayed in IV → VV oxidation processes andcathodic reversible waves from −0.08 to −0.20 V, assignableto the VIV → VIII reduction64 . The cyclic voltammograms obtainedfor the free ligands display irreversible cathodic processes withpotentials that differ from those of the related complexes by ca. 5 mV or less  were observed. Additionally, 1 and 2, which have diethylamine substituents at the phenyl ring. Such a trend is attributed to the highelectron releasing character of the amine group in agreement withthe pattern previously reported for VIV species formedby the phenyl amine (X = NH2)-substituted ONO ligands thatalso display lower potentials than their analogues.65 For 3 and 4, the anodic processesoccur at potentials in the same range (1.1 V vs SCE)of the formerly reported dithiocarbazate species.53 For 1–4, the higher potential cathodicprocess occurs within a short range of potentials (from −0.2to 0.0 V) either for dithiocarbazate or for aroylhydrazone complexes,in agreement with the VIV → VIII reductionbeing less sensitive to the characteristics of the substituents atthe ligand than the VIV → VV oxidation.The electrochemical behavior of tentials 3 than thIV(L1–4)2], i.e. the same species isolated in the solid state, and [VV(L1–4)2]+ and [VVO2(L1–4)]−,derived from the partial oxidation of 1–4.Onthe basis of the results obtained with single-crystal X-ray diffractionand ESI-MS data, the species stable in media containing moderate amountsof water are , [VV(L1)2]+, and [VVO2(L1)]− are depicted in Tables S2–S4. Notably, the prediction of the geometric parametersis very good, as demonstrated by the superposition of the experimentaland calculated structures of complex 1 (The structures of the complexes formed in solutionwere optimized by DFT methods following the procedure reported inthe literature.IV(L1–4)2] (1–4), three differentenergy minima were found, one corresponding to the meridional arrangement of the ligands and two to the facial one. In mer and fac situations, respectively. Notably, for 1 and 2 complexes, the mer isomeris the most stable, while for 3 and 4, itis the fac isomer. This agrees well with the SC-XRDstructures that show the same trend: in fact, the geometry of 1 and 2 is close to the meridional limit and that of 3 to the facial limit. The flexible structureof the ligands accounts for the coexistence of the two isomers. Thefree energy difference between the three isomers is very low, between0.1 and 2.3 kcal mol–1 2]+, the structures are verysimilar to [VIV(L1–4)2], inagreement with the electrochemicalresults that indicate a reversible anodic wave for 1–4, while [VVO2(L1–4)]− are typical dioxidovanadium(V) complexes witha geometry intermediate between the square pyramid and the trigonalbipyramid.Concerning ; the VV species[VV(L1–4)2]+, derivedfrom the oxidation of the previous one, and [VVO2(L1–4)]− were also detected.At these experimental conditions, the non-oxido VIV complexesseem to be stable after 24 h with a slight increase in the oxidationprocess, as indicated by the increment of the relative amount of [VVO2(L1)2]− . This demonstrates that,under the biological experiments, the active species may be one ormore of those present in solution: [VIV(L1–4)2], [VV(L1–4)2]+, and/or [VVO2(L1–4)]−. This was considered during the docking studies.The aged solutions in MeOH/HIV(L1)2] (1), in DMSO and in an aqueous solutionin phosphate buffer at pH 7.4  and mer (1b) isomers. Whenthe spectrum is recorded after 24 h, the pattern remains substantiallyunaltered, even though a significant decrease in the spectral intensityis detected (trace b of IV(L1)2] are clearly visible 2]+ and [VVO2(L1)2]−, as revealed in the ESI-MS experiments described above.The decrease of the signal intensityover time can be related tothe oxidation of Vvideinfra), the binding of complexes may take place close tothe Trp213 residue, this explaining the strong quenching of fluorescenceexperimentally observed.Fluorescence titrationdata for all systemsare depicted in r2) were determined by assumingthat the species present in solution are the complexes [VIV(L1–4)2]; the results are included in 1 and 3 show KSV constants of (1.1–1.2)× 105 M–1, while for 2 and 4,KSV are ca. 2.1 × 105 M–1, confirmingthe high affinity of complexes [VIV(L1–4)2] for BSA. Therefore, the group that seems to influencethe most the strength of the interaction with BSA is the one connectedto the sulfur atom: the replacement of CH3 by CH2–Ph increases twice the quenching constant. This interpretationis also supported by docking simulations.Stern–Volmer quenching constants as wellas the fittingparameter (I0–I)/I] vs log[Q], where Q is the concentrationof complexes 1–4  × 104, are correct, this would indicate a reversible binding of the metalcomplexes to BSA. It must be observed that, considering the existencein solution of [VV(L1–4)2]+ and [VVO2(L1–4)]− besides [VIV(L1–4)2], as suggested by ESI-MS and DFT techniques, a mixture ofthese species may contribute to the affinity of 1–4 to BSA.Thedouble logarithm plot of log[2], derived from the SC-XRD structures and DFT calculations,toward BSA highlight several binding modes with scoring values (Fmax) ranging from 15.5 to 24.5 GoldScore units.Although the scoring values are similar indicating the absence ofbinding specificity, the affinity order mer-[VIV(L2)2] ∼ fac-[VIV(L3)2] > mer-[VIV(L1)2] ∼ fac-[VIV(L4)2] is predicted for thisseries of complexes. The best solutions are located at the interfacesof subdomains IIA/IIIA for [VIV2] (mer-[VIV(L2)2] .Dockingsof the most stable isomers of  11a,c,d aIV(L2)2] 11b. AddiVO2(L1–4)]− show a similar trend, and the affinity orderis [VVO2(L2)]− ∼[VVO2(L1)]− >[VVO2(L3)]− ∼[VVO2(L4)]−, withthe best solutions located at the interfaces of subdomains IIA/IBfor [VVO2(L1)]− (VO2(L2–4)]− (VO2(L3)]− ]− hydrogen bondsare formed between the aza functionality of L1–4 and VV=Ooxido with hydroxyl groupsof Tyr and Thr or NH groups of Arg, Asn, or Lys side chains (Tables S9–S12). For [VVO2(L1–4)]− too, the simulated adductsare in quite proximity of residue Trp213.Docking resultsfor [VO2(L1)]− 12a, IIA/(L2–4)]− 12b,d,anO2(L3)]− 12c. AlsoV(L1–4)2]+, solution analogues to those found forthe [VIV(L1–4)2] are obtained.This is not surprising considering the high geometry similarity betweenthe two series of complexes.Concerning theoxidized species , the oxidationproducts [VV(L1–4)2]+ and [VVO2(L1–4)]− exist in solution, this mixture of species could be responsiblefor the cytotoxic activity of 1–4. As the formation of a small amount of [VVO2(L1–4)]− implies the presenceof an equivalent amount of free ligands in solution, cytotoxicitymay also have the contribution of H2L1–4. However, because all IC50 values are in a rather narrowrange (ca. 6–19 μM) and the amountsof free ligands formed are quite small, we expect the contributionof H2L1–4 to the cytotoxicity to be small.In the light of ESI-MS and DFT results, which suggested that,besides (1–4), the ligands beingtridentate ONS chelating S-alkyl/aryl-substituted dithiocarbazatecompounds. Three of these complexes, [VIV(L1–3)2] (1–3), were analyzedby single-crystal X-ray diffraction studies, revealing that the coordinationgeometry around the V center is distorted octahedral (1 and 2) or trigonal prismatic (3). Additionally,considering the formed O2N2S2 coordinationenvironment around the VIV center, 1 and 2 correspond to the mer isomers, while in 3, a fac arrangement is formed. Interestingly,EPR data and DFT calculations indicate the coexistence of mer and fac isomers in solution, with comparablestability; probably, subtle factors like the solubility and/or thecrystal packing may determine the isolation of one of the two isomericforms in the solid state. To the best of our knowledge, this is oneof the first studies in which the mer and fac isomers of non-oxido VIV species are in equilibriumin solution, and this possibility should be considered in the futurefor the characterization of similar compounds.In this work,we report the preparation and characterization inthe solid state and in solution of a group of new mononuclear non-oxidoVIV(L1–4)2] complexes are quite stable in solution for severalhours, but suggested their partial oxidation to [VV(L1–4)2]+ and [VVO2(L1–4)]−, these changesproceeding faster as the relative amount of water and time increase.It is worth notingthat EPR, UV–vis, and ESI-MS data allowedto confirm that the obtained [VIV(L1–4)2]with proteins is relevant; this was studied with BSA by fluorescencequenching and computational methods. Docking calculations revealednon-covalent interactions with different regions of BSA, particularlywith Tyr, Lys, Arg, Asn, and Thr residues. On the one hand, they allowedto confirm the fluorescence quenching results, since the adducts formedare found close to Trp213 residue; on the other hand, they highlightedthat [VIV(L1–4)2], [VV(L1–4)2]+, and [VVO2(L1–4)]− interactwith different strengths and different subdomains of the protein,depending on the vanadium species and on the ligand coordinated.Since we focus on the use vanadium compounds as pharmaceuticals,namely, for cancer treatment, the understanding of the interactionof complexes , oxidation/hydrolysiscertainly takes place to some extent during the experiments in water.Therefore, a mixture of VIV, VV, and VVO2 species, being present in these systems, could be responsiblefor the activity observed. The small amounts of free ligands formedmay also contribute. These results reinforce our recent conclusionsthat, regardless of the oxidation state of one of these administeredvanadium drugs, a mixture of oxido and non-oxido VIV andVV species could be formed at the physiological conditions,their relative amount depending on the nature of the ligand and thermodynamicstability of the initial compound; therefore, the biological actionshould be interpreted and explained assuming the existence of morethan one V species in solution, probably in the +IV and +V oxidationstates.Concerning their possible use as anti-cancer agents,