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,


Designing new medical devices with advanced humidity sensors is of great significance for patients with incontinence-associated dermatitis (IAD). The primary goal of this study is to test the humidity-sensing mattress system for patients with IAD in clinical settings. The design of the mattress is set at 203 cm, with 10 × 3 sensors, dimensions of 19 × 32 cm, and a weighted bearing of 200 kg. The main sensors consist of a humidity-sensing film, a thin-film electrode (6 × 0.1 mm), and a glass substrate (500 nm). The sensitivity of the test mattress system showed that the resistance-humidity sensor was at a temperature of 35 °C ( Medical mattresses with advanced humidity sensors are receiving significant attention when it comes to treating patients with IAD ,2,3. Des devices ,5,6. Som devices , skin te devices , metal o devices , transep devices , and wat devices . In the  devices ,13,14, f devices , and the devices .Humidity sensors for patients with IAD have been tested with moisture-associated dermatitis, liquid, and water vapour ,8,10. ThTypically, the traditional design of humidity-sensing mattresses for patients with IAD has limitations, such as early IAD diagnosis ,7. IndeeThus, there still remains a lack of advanced humidity sensors which have been tested with patients exhibiting early signs of IAD. Indeed, the characteristics of sensing moisture-associated skin damage are limited, focusing on different types of sensing materials . PreviouAccordingly, our study has designed a humidity-sensing mattress for patients with IAD following exposure to experimental testing in clinical settings. Firstly, the design of the mattress consists of a humidity-sensing film, a thin film electrode, and a glass substrate. Secondly, the design is also focused on the stability of the humidity sensors , ambient temperature, and adsorption–desorption dynamic cycles. Finally, when testing the response time of 10–120 s with five volts, the magnitude ranged from 1The humidity sensor is real-time monitoring of wetting moisture, water vapour, and vibration urine for converting the data into a corresponding electrical signal;The thin-film electrode array is a method for the fabrication of submicrometer gold on polyester and silicon. The sensor structure consists of a pair of interdigital electrodes and aluminium films on the glass substrate for the thickness (500 nm).When it comes to the sensing design of the mattress environmental chamber (MEC), it tests individual weight-loading support of 200 kg. The MEC consists of a lengthwise, zipped opening to allow mattress placement, as well as a loading array. The design of the testing mattress has a set of 203 cm × 96.5 cm peripheral outlets and a moisture-bearing patient moisture reservoir (PMR). The PMR rests on humidity sensors consisting of 0.28AH), parts per million (PPM), and the dew/frost point (D/F PT). The humidity sensor classifies the mass of water vapour, in grams per cubic metre, and feet (1 grain = AH is the g/3 or ft3). The RH is tested as the ratio of moisture to the maximum level, which gives the temperature and humidity. In the following, RH is equated as:SH) is defined as:SH is the g/SH is the temperature in a unit volume of gas. RH calculates the ratio of AH to SH as follows:A humidity sensor tests the amount of water vapour and temperature . It is wthe water vapour. The PPMv and PPMw are amongst the AH, the D/F is based on the temperature (above 0 °C), and PT is the temperature (below 0 °C). However, the D/F point parameters are the AH.The PPMv is defined as the volume of water vapour in dry gas, the PPMw refers to of voltage (The sensitivity of humidity is determined with the fixed capacity The sensing sensitivity (S) is defined using the following equation:Shimizu et al.  tested tA porous The humidity-sensing mattress is designed for clinical settings: 203 × 96.5 cm, 10 × 3 sensors, and a dimension of 19 × 32 cm . The phyIn the humidity-sensing mattress, the recording functions include system setup, data storage, graphing, and an alarm. Sensitivity is performed using a system calibration with preferences regarding the humidity sensor. Data storage is recorded as historical records and current data. The graphing function allows the patients and their caregivers to observe the recording. The sensor will present a signal on the screen if a hazard over the threshold is detected. The flowchart of the humidity-sensing system is shown in In the warm-up system, with regard to the humidity-sensing mattress platform, there is a connection between ScreenView and the processor . The response time is set at 1 min at 100 Hz on Arduino technical data. Analogue inputs of the Arduino board are used to extract data at FSR400. The mapping of the contact point of the humidity-sensing platform is shown in The testing of long-term bedridden patients with IAD is arranged with 19 volunteers of different temperature and humidity datasets. All volunteers are tested from the same number of patients with different values of BMI and pH index see . To ensuA total of 19 voluntary patients with IAD underwent experimental testing at the Thammasat University Hospital, Thammasat University Rangsit Campus, Pathum Thani, Thailand. All patients provided informed consent to participate in the study. The Human Research Ethics Committee of Thammasat University (Science), Thailand, approved the study project in accordance with compliance with the Declaration of Helsinki, the Belmont Report, CIOMS guidelines, and international practice (ICH-GCP) (COA 032/2566 Project No. 66NU016). The body position regions consist of three sensors in patients with IAD: sensor 1 is the head and neck , sensor 2 is the shoulders (rhomboid major muscles), and sensor 3 is the lumbar spine (spine endpoint), as can be seen in We designed the mattress so that it consists of a humidity sensor on the surface of the substrate and electrodes. A thin-film electrode is built on the glass substrate, which detects thickness at 500 mm. The length and width of the thin-film electrodes are approximately 0.1 mm, with two substracts (10 × 4 mm), as can be seen in The experimental data show that SHT10, DS18B20, and FSR 400 are inputs, based on the mean values of the recorded data. The aqueous solutions of humidity sensors for long-term bedridden patients with IAD are set as LiCI, MgCThis principal part of the sensor is nodulised under a vacuum ranging from 30–100 °C for 30 min, with the voltage signal of 0.85 V for the same RH change. The sensor resistance is extracted from 10 cycles, and the humidity is changed to 5% HR with a 10 s change ratio at a constant temperature of 35 °C. The resistance-humidity characteristics of the sensor are 35 °C, voltage  are repeatedly tested with calibration changes at 0.5% RH, voltage , which achieved 90% of the total impedance change. The sensitivity exhibited a fast response and a recovery time of between 10 s and 30 s, which is the best reported time for a resistive type of humidity-sensing mattress for IAD in clinical settings ,32,37. Ag et al. , Bian etg et al.  and Chu g et al.  fabricatIn conclusion, the design of the humidity-sensing mattress has been tested with regard to the water vapour with RH, AB, PPM, and D/F PT. The testing showed resistance-humidity characteristics of the sensor at 35 °C (The development of the humidity-sensing mattress is a crucial step in the production of a novel piece of medical technology that provides a faster response, high sensitivity, and recovery time; indeed, it may also be a potential design for practical application. Notably, the design of the humidity-sensing mattress compounded with the thin-film electrode as the RH increased to 90%, showing excellent sensitivity. This property enables the sensor to be applied to medical diagnosis for patients with IAD. When the design has been applied, and the enhanced clinical acceptance of sensing material is a reality, it will be possible to provide and develop a low-cost mattress for IAD in clinical homecare. Once available, this humidity-sensing mattress has broad application prospects in the field of flexible sensors, wearable medical diagnostic devices, health detection, etc.


This complex is very stable toward dioxygen due to steric protection of the nickel(II) center. Here, we report the dioxygen reactivity of a new Ni–NO complex, [Ni(NO)(I)(L1″)], with a less hindered N2 type bis(pyrazolyl)methane ligand, which creates a coordinatively unsaturated ligand environment about the nickel center. Here, L1″ denotes bismethane. This complex is also described as a hs-nickel(II) center with a bound 3NO−, based on spectroscopic and theoretical studies. Unexpectedly, the reaction of [Ni(NO)(I)(L1″)] with O2 yielded 2-O2N)(L1″)2](I3) (L3 SNO = 1/2) to 1NO+ , and to NO−  when binding to a transition metal ion, i.e., Mn+–NO·, Mn−1–NO+, and Mn+1–NO−  complex.Nitrogen monoxide (·NO) is a small, relatively unstable, potentially toxic, diatomic free-radical molecule. ·NO has become one of the most studied and fascinating molecules in biological as well as inorganic chemistry ,7,8,9,10Mn+1–NO− ,13. By cMn+1–NO− . In the 6F5)(L1)]  ,16. This1)] − in ), and it1)] − in ,16. We h1)] − in . Later, omplexes ,10 with omplexes , Co 21]6F5)(L1)]omplexes , and Cu omplexes . Using dectively ,21,22. T· ligand ,10,23.10 system, did not show any reactivity toward dioxygen  and [Cu(κ1-ONO)2(L1″)]. The main differences between these complexes relate to the different ligand charges  and bulkiness (tertiary butyl versus isopropyl substituents)  and [CuCl2(L1″)], which again differ for the same reasons mentioned above  (PF6)       , an hs-{NiNO}dioxygen . This isdioxygen   . We alsoed above ,26. Thised above , [Ni(NO)ed above  (L0f− = NO)(L0)] ,29 (L0− n)](PF6)  (iPr3tacmp-tol)]  (Tmp-tolTseMes)]  (TseMes−BuIm)3}]  (HB(tBuIH2Im)3}]  (PhB(CyCMeBz)3}]  (HB(MeBzPhTz)3}]  (HB(p-tB3 group [[Ni(NO)(I)(L1″)] with a less hindered N2 type bis(pyrazolyl)methane coligand to create a coordinatively unsaturated ligand environment. We obtained an unexpected, six-coordinate compound, 2-O2N)(L1″)2](I3)[Ni(L3)], [Ni(NO)(L0f)] is also stable toward dioxygen due to the hindered CF3 group . In this3)(PPh3)2] was reported in 1971 by Professor Enemark , was obtained using [Ni(NO)(Br)(PPh3)2] as the precursor [3)2] with the potassium salt of the ligand [n could also be used for the synthesis [2(I)]2 and [Co(NO)2(I)]n were used to synthesize M–NO complexes such as [Fe(NO)(L3)] [2(L1″)](BF4), and [Co(NO)2(L1″)](BF4) [3)2] and [Ni(NO)(I)]n [n as starting material in this research. The Ni–NO complex [Ni(NO)(I)(L1″)] was obtained by the reaction of [Ni(NO)(I)]n and the ligand L1″, as shown in [Ni(NO)(I)(L1″)], suitable for structural characterization. The coordinated iodide ion is needed for the structural stability of the complex. If we add a THF solution of AgPF6 to a solution of [Ni(NO)(I)(L1″)] to remove the iodide ion, the color of the solution changes from green to colorless, indicating that, upon removing the iodide, the complex decomposes.The first structural characterization of a Ni–NO complex [Ni(NO)(L3)] , [Fe(NO)″)](BF4)  previousNO)(I)]n . Therefo[Ni(NO)(I)(L1″)] is shown in The structure of [Ni(NO)(I)(L1″)] is four-coordinate with three nitrogen atoms from L1″ and NO and the iodide bound. The Ni1–N11 and Ni1–N21 bond lengths (2.0157 (12) and 2.0234 (12) Å) are experimentally equivalent and considerably longer than the Ni1–N1 (1.6467(16) Å) bond. The N1–O1 bond length is 1.136 (3) Å and the Ni1–N1–O1 angle is 172.42(17)°. The presence of the bidentate ligand, with a restricted bite angle, introduces significant distortions in this coordination geometry. Thus, the variation in the N–Ni1–N angles is substantial, with the narrowest angle being 91.41 (5)° for N11–Ni1–N21 and the widest being 123.86 (6)° for N1–Ni–N11. The value of τ4 = [360 – (α + β)/141] = 0.80, where α and β are the two widest angles subtended at the metal center [τ4 in [Ni(NO)(I)(L1″)] is closest to 0.85, which corresponds most closely to a trigonal pyramidal geometry (C3v). In this structure, the three nitrogen donors form the basal  plane. To investigate this further, we calculated the distances between the basal planes formed by three of the coordinating atoms and the nickel center. Here, the distance between Ni1 and the plane formed by N11, N21, and I1 is much longer (1.0817 (3) Å) compared to the other three: 0.4900 (3) Å between Ni1 and the plane formed by N11, N21, and N1; 0.5673 (3) Å between Ni1 and the plane formed by N11, N1, and I1; and 0.5493 (3) Å between Ni1 and the plane formed by N21, N1, and I1. The latter three distances are quite similar. Based on these considerations, the coordination geometry of the complex is distorted tetrahedral, which is a different conclusion than the result from the τ4 value discussion.The coordination geometry of Ni in l center . The val−1 in these compounds, showing a larger degree of variability and indicating distinct differences in the electronic structures of these complexes. Therefore, careful spectroscopic experiments as well as theoretical support are very important to describe the true electronic structure of [Ni(NO)(I)(L1″)], as well as the oxidation state of the metal center and the NO group  were measured using KBr and CsI pellets, respectively (I)]n (−1 in [Ni(NO)(L0f)] [−1 in [Ni(NO)(L3)] [−1 in [Ni(NO)(I)(L1″)], as listed in −1, and is assigned considering related values, 574 cm−1 in [Ni(NO)(L3)] [−1 in [Ni(NO)(L0f)]  [IR and far-IR spectra of ectively . The ν(I)]n . ConsideO)(L0f)]  > 1780 cNO)(L3)]  > 1777 cNO)(L3)]  and 598 O)(L0f)]  (L0f)] .[Ni(NO)(I)(L1″)] are shown in d–d transitions [6F5)(L1)], the band at 804 nm is assigned to the 3T1(F) → 3T1(P) transition, giving rise to a pseudo A signal in MCD (magnetic circular dichroism) because of instate spin–orbit coupling [[Ni(NO)(I)(L1″)] exhibits strong absorption bands in the 500–700 nm range, as shown in d–d transitions of the high-spin Ni(II) center. Therefore, the UV–Vis absorption data indicate that the Ni oxidation number is +II, and that the Ni(II) center is in the high-spin state. This implies that the NO ligand is reduced in the complex to 3NO− (nitroxyl), since the complex charge is zero. This is further discussed in the DFT section. The complex [Ni(NO)(I)(L1″)] has a green color {λmax/nm (ε/M−1cm−1): 492 (130) and 717 (400)}, whereas [Ni(NO)(L3)] {λmax/nm (ε/M−1cm−1): 630 (480)} [λmax/nm (ε/M−1cm−1): 593 (440)} [[Ni(NO)(I)(L1″)] versus [Ni(NO)(L3)]), causing a weaker overall ligand field and a shift of the 3T1(F) → 3T1(P) type d–d transition to lower energy in [Ni(NO)(I)(L1″)] (717 nm) compared to [Ni(NO)(L3)] (630 nm) and [Ni(NO)(L0f)] (593 nm). These spectral features are different from those of the starting material [Ni(NO)(I)]n (UV–Vis spectra (dichloromethane solution and solid state) of nsitions . In [Ni}  and [Ni(3 (440)}  are blueNO)(I)]n .1H and 13C) are compiled in [Ni(NO)(I)(L1″)] has relative sharp 1H-NMR signals in both CDCl3 and (CD3)2CO solution, as shown in [Ni(NO)(I)(L1″)] is +II, and the metal center is in the high-spin state. Therefore, the total spin of the Ni(II) center should be S = 1. However, the NMR spectra clearly indicate that the complex is diamagnetic . This originates from antiferromagnetic coupling between the d-electrons of Ni(II) and the π*-electrons of the nitroxyl ligand . This assignment was previously proposed for other complexes, listed in Nuclear magnetic resonance (NMR) spectra ([Ni(NO)(I)(L1″)], on the other hand, reacts with dioxygen at room temperature ([Ni(NO)(I)(L1″)] and dioxygen are shown in [Ni(NO)(I)(L1″)] slowly reacts with dioxygen. We isolated the product and grew crystals. Crystallographic data of the product show that an unexpected six-coordinate structure was obtained, with the oxidation of 3NO− and I− to NO2− and I3−, as shown in [Ni(NO)(I)(L1″)] decomposed, and L1″ itself was reconstituted. The product complex has a six-coordinate structure with two L1″ ligands bound to the nickel ion and one coordinated NO2−, which originates from the oxidation of 3NO−. The complex is paramagnetic, as evident from 1H-NMR spectroscopy, and therefore contains a hs-Ni(II) center.As mentioned above, the coordinately saturated Ni–NO complexes [Ni(NO)(L3)] and [Ni(NO)(L0f)] do not react with dioxygen. The coordinately unsaturated Ni–NO complex perature . During 2-O2N)(L1″)2](I3)(I3)(I3)[Ni stretches of the coordinated NO2− group (s(N–O) stretch of the NO2− group in [Cu(κ2-ONO)(L3)] was observed at 1264 cm−1 (I3)[Ni] ]  (I3)[Ni(L1″)2](I3) complex using def2-TZVP as the basis set and different functionals. The results are shown in 10, but for other {MNO}x systems as well , we therefore investigated whether the analogous open-shell singlet electronic state could be geometry optimized. Excitingly, using B3LYP we were able to obtain a fully optimized structure of the complex in the BS state. The resulting wavefunction shows spin densities of +1.05 on Ni(II) and −1.19 on the NO− ligand. A superposition of the calculated BS structure and the crystal structure, shown in In these calculations, a closed-shell (CS) electron configuration was used. However, experimentally, the related complex [Ni(NO)(L3)] has been shown to have an open-shell ground state, with a hs-Ni(II) center  . Neverth[Ni(NO)(I)(L1″)] in the BS state is about 2 kcal/mol lower compared to the CS system. Given the similarities between the structural and spectroscopic properties of [Ni(NO)(I)(L1″)] and [Ni(NO)(L3)] and the detailed investigations on the latter complex [[Ni(NO)(I)(L1″)], as observed for [Ni(NO)(L3)]. Attempts to geometry-optimize the complex in the BS state using BP86 were not successful, as the wavefunction always collapsed into the CS state. Comparing the results obtained for the open-shell (BS) and CS systems using the B3LYP functional, we see that for the BS state, the Ni–NO bond distance is longer and ν(Ni–NO) is lower compared to experiment. In contrast, for the CS state, the Ni–NO distance is underestimated and ν(Ni–NO) is overestimated. These results show that the experimental Ni–NO bond strength is in between the predictions for the open-shell and closed-shell system. On the other hand, the Ni–N–O bond angle and the N–O stretching frequency obtained for the BS state are closer to the experimental values. The calculated energy for  complex , these r2-O2N)(L1″)2]+[Ni(I)(L1″)] with O2. In this case, the complex does not involve an antiferromagnetically coupled ground state. For this reason, optimization and frequency calculations were performed using the functional BP86, since it shows the overall best results for the Ni–NO complex. The results from these calculations are shown in In order to assist with the experimental findings, DFT calculations were also performed for the −1, which shift to 1247/1241 cm−1 upon 18O isotope labeling . On the other hand, the antisymmetric N–O stretch of coordinated nitrite is predicted at 1167 cm−1, shifting to 1147 cm−1 with 18O. The experimental value for the N–O stretch is observed at 1201 cm−1, which is right in between those predicted frequencies. However, the experimentally observed isotope shift of 19 cm−1 , and NO gas was purified by passing through a column filled with solid NaOH before the reaction. 18O2 gas was obtained from Shoko Science and was used without any purification . Other reagents were commercially available and used without further purification. The bismethane (L1″) was prepared following a published method  was obtained from crystallographic data by removing the tetrahydrofuran molecule present in the structure. The initial structure for 2-O2N)(L1″)2][Ni [[Ni(NO)(I)(L1″)], we performed both closed-shell and broken-symmetry calculations.Computational studies were performed using Gaussian 16 software . The geof2-TZVP) ,49. The f2-TZVP)  and the f2-TZVP) ; (ii) Bef2-TZVP) , along wf2-TZVP) . For 19H32IN5NiO·0.5(H2O): C 42.89, H 6.06, N 13.16. Found: C 41.94, H 5.87, N 12.77. IR (KBr/cm−1): 2965 s, 2933 m, 2869 s, 1776 vs ν(N–O), 1547 m, 1461 m, 1437 m, 1402 w, 1382 m, 1366 w, 1281 s, 1181 m, 1053 m, 809 m, 796 m. Far-IR : 661 s, 571 m, 543 w, 470 w, 410 w δ(Ni–N–O), 381 m, 329 w, 303 w, 252 w, 212 w, 188 w, 163 w. 1H NMR (CDCl3) δ/ppm (assignment): 1.25 2), 1.40 2), 1.67 2), 2.75 2), 3.83 2), 4.02 , 6.24 . 1H NMR (acetone-d6) δ/ppm (assignment): 1.30 3), 1.47 3), 1.71 3), 3.23 3), 3.85 3), 3.95 , 6.60 . 13C NMR (CDCl3) δ/ppm (assignment): 22.9 (CH(CH3)2), 25.7 (CH(CH3)2), 29.2 (CH(CH3)2), 53.1 (H2C), 100.6 (pz-4C), 152.4 (pz-3C), 164.7 (pz-5C). UV–Vis  λmax/nm (ε/M−1cm−1): 290 (4060), 375 (340), 492 (130), 717 (400). UV–Vis  λmax/nm: 297, 383, 500, 646, 730.Anal. Calcd for C[Ni(NO)(I)(L1″)]  was dissolved in dichloromethane (9 mL) at room temperature in an argon atmosphere. The argon was then replaced with O2 gas and the solution was stirred at room temperature for 3 days. During this time, the color of the solution changed from deep green to brown. Recrystallization from dichloromethane/n-heptane at −30 °C gave brown crystals. Single crystals suitable for X-ray diffraction were obtained by slow recrystallization under the same experimental conditions. Yield: 45% . In a 50 mL Schlenk tube, 38H64I3N9NiO2: C 40.81, H 5.77, N 11.27. Found: C 40.67, H 5.77, N 11.27. IR (KBr)/cm−1(assignment): 3123 w, 2967 vs, 2930 s, 2870 m, 1549 s, 1475 s, 1459 s, 1442 s, 1383 s, 1366 m, 1336 w, 1279 vs, 1201 s(ν(NO2)), 1183 s, 1055 m, 1016 w, 812 w, 661 w. Far-IR (CsI) cm−1: 672s, 662 s, 584 w, 539 m, 508 w, 475 w, 413 w, 391 w, 320 w, 311 w, 228 w, 182 w, 138 vs. UV–Vis  λmax/nm (ε/M−1cm−1): 295 , 365 . UV–Vis  λmax/nm: 301, 376.Anal. Calcd for C18O2 reaction, [Ni(NO)(I)(L1″)]  was dissolved in dichloromethane (5 mL) at room temperature in an argon atmosphere. The argon was then replaced with 18O2 gas and the solution was stirred at room temperature for 3 days. After the reaction, the recrystallization was carried out using the above procedure.For the −1(assignment): 3125 w, 2968 vs, 2931 s, 2870 m, 1550 m, 1473 m, 1459 m, 1441 m, 1385 s, 1366 m, 1335 w, 1280 vs (ν(N18O16O)), 1182 m, 1095 w, 1079 m, 1055 m, 1018 m, 810 m, 661 w.IR (KBr)/cm[Ni(NO)(I)(L1″)] and 2-O2N)(L1″)2](I3) with the less hindered N2 type bis(pyrazolyl)methane coligand L1″, to create a coordinatively unsaturated ligand environment, where L1″ denotes bismethane. The coordination geometry in [Ni(NO)(I)(L1″)] is four-coordinate with three nitrogen atoms from L1″ and NO and a bound iodide anion. We could conclude that the coordination geometry about the Ni center is distorted tetrahedral, based on distances between the basal planes and the metal ion. The ν(N–O) value is 1777 cm−1 in [Ni(NO)(I)(L1″)], which is clearly shifted from ν(N–O) in the starting material [Ni(NO)(I)]n, observed at 1854 cm−1. Moreover, the Ni–NO stretching vibration, ν(Ni–NO), is observed at 572 cm−1. This assignment is based on the consideration of the vibrational frequency of ν(Ni–NO) in related complexes, especially [Ni(NO)(L3)]. Considering the electron-donating properties of hydrotris(pyrazolyl)borate type ligands, the overall electronic structure of the related complexes [Ni(NO)(L3)] and [Ni(NO)(I)(L1″)] is almost the same, in spite of the different coordinating groups (pyrazolyl nitrogen versus iodide anion) and different ligand charges . From the UV–Vis assignments, the Ni oxidation state is +II, and the Ni(II) center is in the high-spin state. This implies that the NO ligand is 3NO− (nitroxyl) in the complex, since the complex charge is zero, which is analogous to the electronic structure reported for [Ni(NO)(L3)]. In comparison, [Ni(NO)(I)(L1″)] has a green color, not blue like [Ni(NO)(L3)]. This difference relates to the different ligand donor set (N3I1 versus N4), causing a shift in the low-energy d–d transitions in [Ni(NO)(I)(L1″)].Previously, we reported a Ni–NO complex with a hindered N3 type coligand, [Ni(NO)(L3)], where L3l)borate . This co[Ni(NO)(I)(L1″)], we obtained an unexpected six-coordinate compound, 2-O2N)(L1″)2](I3)[Ni(I)(L1″)] to O2, which can be attributed to one of the ν(N–O) modes of the bound NO2− group. For the anion I3−, the νas(I–I) band was observed at 137 cm−1, and characteristic strong bands in the UV region of the absorption spectrum were observed at 295 nm  and 365 nm , which are assigned as σ → σ* and π → σ* transitions of I3−, respectively.After dioxygen reaction of [Ni(NO)(I)(L1″)] using both closed-shell and broken-symmetry electronic states were conducted using different DFT methods. Notably, using B3LYP, we were able to optimize the structure of the complex for both of these electronic states, allowing us to make direct comparisons. The results suggest that the complex is best described as a hs-Ni(II) center (S = 1) antiferromagnetically coupled to the 3NO− ligand , with the broken-symmetry state being about 2 kcal/mol lower in energy than the closed-shell state. The electronic structure of [Ni(NO)(I)(L1″)] is therefore analogous to that of [Ni(NO)(L3)] reported previously, in agreement with their very similar structural and spectroscopic properties. DFT calculations for the six-coordinate product complex 2-O2N)(L1″)2]+[Ni(κ were also performed. Our results suggest that the N–O stretching mode of nitrite, observed experimentally at 1201 cm−1, corresponds to the antisymmetric stretch, predicted at 1167 cm−1 with a calculated 18O isotope shift of about 20 cm−1, in agreement with experiment.Theoretical calculations for


The deuterium dose-to-mother (DTM) method measures the human milk intake of breastfed children. Recently, the use of this method has been expanded to classify babies objectively as exclusively breast fed (EBF) or not (non-EBF) based on quantification of non-milk oral water intake (NMOI). However, the calculation of NMOI estimates involves atmospheric temperature and humidity.To evaluate the effects of atmospheric temperature and humidity on NMOI calculation and the classification of exclusive breastfeeding.The effect of indoor temperature and relative humidity on NMOI and the estimated prevalence of non-EBF were examined in two existing data sets of DTM in children by varying temperature in the range of 15 to 35°C and relative humidity in the range of 20 to 80% representing the maximum span of indoor conditions expected. Population-level estimates of NMOI distributions were derived using the rstan package for R v2.21.2.The NMOI decreased at a rate of −1.15 g/day per °C increase and at a rate of −1.01 g/day per percent increase in relative humidity; this was due to variations in non-oral water intake from the atmosphere, a component of the calculation of NMOI, which is dependent on temperature and humidity. For the various locations considered, the mean calculated NMOI varied between 24.6 and 53.3 g/day using the same input data. In the mixed-fed sample of babies, the prevalence of non-EBF based on the earlier defined NMOI cut-off of 86.6 g/day was reduced by 19% when relative humidity was increased by 60%.Atmospheric conditions are essential factors in the computation of NMOI, used in the objective classification of babies as exclusively breast fed or not, and should be considered when the DTM method is used to classify exclusive breastfeeding. The However, assumptions were made in calculating NMOI using the DTM technique . The firTo the best of our knowledge, there have been no systematic evaluations of the influences of atmospheric temperature and humidity on the constants used in the calculation of NMOI. Therefore, in this study we computationally examined the effect of varying atmospheric temperature and relative humidity on calculating NMOI using the DTM method, which in turn is used in the objective classification of babies as exclusively breast fed or not.2.The DTM method of estimating breast milk transfer from mother to baby involves collecting a series of saliva (body water) samples from the mother and baby over two weeks. The DTM method is based on a two-compartment model first described by Coward et al.  as shownbm, kg/day); the fractional water turnover in the mother  and the total water loss in the baby  are estimated. Breast milk intake and Non-Milk Oral Intake estimates are further estimated using these parameters and few assumptions.Four parameters are estimated by minimizing the difference between observed and fitted deuterium enrichment values for mother and baby combined. Parameters namely: Deuterium enrichment in the mother's body water at time zero , the transfer of water from the mother to the baby via breast milk (F2.1.The component of interest in this study was the NMOI (kg/day), which can be estimated from Total water output, Water for growth, Intake of water from breast milk and Non-oral water intake .(1)NMOI=2.2.rcE g/day) was calculated from the rate of efflux (rbE = 0.105 g/min/m2) and body surface area (BSA) (m2).2) of the child is given by n (FRAC) .(2)Fob=FHRexp, %), is 94.6% at temperature of expired air W, kg) (The rate of respiratory water efflux (g/day) was calculated as the product of inspired air volume  and absolute humidity  .IAV=RIA×rtE, g/day)   is given2.3. formula , 10 TBW of a baby is the atmospheric water absorbed through the skin of the baby and the respiratory water influx through the lungs; this was estimated as the product of the proportion of non-oral water intake from the atmosphere (NOWIA) and the total water intake of the baby (rbl- g/day) is calculated as the product of IAV (IAV- L/min) and absolute atmospheric humidity (mg/L)  wasHR(%) is the relative atmospheric humidity, and T(°C) is the atmospheric temperature. The transcutaneous water influx (rcl- g/day) was calculated as 2, HA-absolute humidity (mg/L) ASAT- the atmospheric saturation (mg/L) and BSA (m2) -the BSA of the baby. Therefore, the influx correction wasThe non-oral water intake (the baby .(5)Fa=NOy (mg/L) . rbl=IAVM method . These c2.6.n = 113, body weight 4.0 kg to 7.8 kg) who were observed and verified as EBF  of EBF babies in the Indonesian study was used  , NOWIA iConsequently, the NMOI decreased by −1.2 g/day per °C increase  and relaUsing published average annual indoor temperature and relative humidity across different geographic locations , 13, theThe estimated NMOI (mean and standard deviation) in a sample of 113 EBF Indonesian babies  under va3.2.n = 221) (Under standard IAEA assumptions  and usinn = 221)  were clan = 221) .4.This study examined the effects of atmospheric temperature and relative humidity on calculating NMOI using the DTM method. The assumed NOWIA increased with increasing temperature and relative humidity, resulting in an approximately 1 g/day decrease in the NMOI per unit increase in atmospheric temperature and relative humidity.The DTM technique was initially developed to measure breast milk intake in babies; indeed, the accuracy of the computation of breast milk intake was intact and unaffected by temperature and relative humidity; this reinforces confidence in the technique used to quantify breast milk intake. However, several studies , 15 haveFurthermore, it casts doubt on using cut-off-based deterministic identification of the EBF. The variation in proportion of children classified as EBF based on different temperatures and humidity demonstrates the possibility of misclassification if atmospheric temperature and humidity are not considered in the NMOI. A probability-based approach that assigns the probability of being EBF to every baby based on the distribution of NMOI in the standard EBF population would be better. However, adjustment for atmospheric temperature and relative humidity remains essential to obtain the correct distribution of the NMOI, even when using the probability-based approach. Liu et al.  clearly Atmospheric relative humidity had a more significant impact on the NMOI than temperature. When the relative humidity was increased by 60%, and the temperature was held constant, the prevalence of non-EBF, based on the previously defined NMOI cut-off of 86.6 g/day, was reduced by 19%. Ideally, the cut-off would be constant across geographical locations for the temperature-and relative humidity-adjusted NMOI.To the best of our knowledge, this is the first study that closely examines, although theoretically, the implications of variations in atmospheric conditions on the computation of NMOI, which is becoming an increasingly important output of the DTM technique. However, a simple correction cannot be used to account for variations in temperature and relative humidity in the calculation of NMOI because of the complex calculations involved for NMOI. A web-based, user-friendly application could incorporate the actual atmospheric temperature and relative humidity, thus providing more accurate NMOI calculations. However, this would require a predictable association between meteorological data and indoor atmospheric conditions. This association needs to be understood to easily incorporate these adjustments in the NMOI calculations in the future. The calculation of NMOI can also be affected by the variation in atmospheric temperature and humidity that the baby is exposed to, during the DTM study period of 14 days, and this needs to be explored. To improve the accuracy of the estimation of NMOI, measurement of atmospheric conditions in the baby's immediate environment could be added to future protocols. However, the gain in accuracy must be balanced against the increased complexity of the method in the field as well as in the calculations. A limitation of the study is that the variation in NMOI presented in this paper is a theoretical demonstration and has not been validated in field setting.In conclusion, this study demonstrates the need to consider atmospheric temperature and relative humidity when calculating NMOI.