Positron emission tomography (PET) is a non‐invasive imaging method to measure the molecule in vivo. PET imaging can evaluate the central nervous system drugs as target engagement in the human brain. For antipsychotic drugs, adequate dopamine D2 receptor occupancy (“therapeutic window”) is reported to be from 65%‐70% to 80% to achieve the antipsychotic effect without extrapyramidal symptoms. For antidepressants, the clinical threshold of serotonin transporter (5‐HTT) occupancy is reported to be 70%‐80% although the relation between the side effect and 5‐HTT occupancy has not yet been established. Evaluation of norepinephrine transporter (NET) occupancy for antidepressant is ongoing as adequate PET radioligands for NET were developed recently. Measurement of the target occupancy has been a key element to evaluate the in vivo target engagement of the drugs. In order to evaluate new drug targets for disease conditions such as negative symptoms/cognitive impairment of schizophrenia and treatment‐resistant depression, new PET radioligands need to be developed concurrently with the drug development. 11C]raclopride for dopamine D2 receptors decreased from (A) baseline to (B) antipsychotic administration conditions.PET imaging can evaluate the central nervous system drugs as target engagement in the human brain. The uptake of [ PET imaging can evaluate biodistribution and target engagement in the human brain of central nervous system (CNS) drugs.22.118F]fluorodeoxyglucose ([18F]FDG), which can measure the glucose metabolism, accumulates in tissues of high glucose consumption such as tumor and inflammation, and produces the large amount of gamma radiations there. By detecting the radiation, the location of the tumor and inflammation is identified.PET radioligands are, in most cases, administered to the human intravenously. The injected PET radioligand is distributed according to its characteristics such as blood‐brain barrier (BBB) permeability, affinity for target molecule, metabolism, and exertion from body The positron is emitted by positive beta decay and converts to two gamma radiations by annihilation to electron. These gamma radiations are measured outside the body using PET machine. Consequently, the degree of targeted molecule of each PET radioligand can be evaluated quantitatively. For example, [2.218F]FDG has been the most used PET radioligand, especially in oncology field. For [18F]FDG‐PET, visual inspection and semi‐quantitative measurement using standardized uptake value (SUV) are useful for clinical evaluation. However, in most cases of target engagement in the brain, more accurate quantitative analysis is necessary for the evaluation of PET radioligands. When a new promising PET radioligand is developed, quantitative analysis is made to establish the most appropriate method for the PET radioligand.raclopride is the PET radioligand, which selectively binds to dopamine D2 receptors.11C]raclopride can measure dopamine D2 receptors in the striatum in the brain due to moderate affinity to the receptor. For the evaluation of extrastriatum, where the density of dopamine D2 receptor is quite low, [11C]FLB45718F]fallypride11C]PHNO has the affinity for dopamine D3 receptor as well as dopamine D2 receptor.11C]PHNO reflects the dopamine D3 receptors in the regions which has relatively higher density of dopamine D3 receptors than dopamine D2 receptors such as globus pallidum.Main mechanism of action of antipsychotics has been the inhibition of dopamine D2 receptors. Craclopri4.2Farde et al reported the striatal dopamine D2 receptor occupancy of several antipsychotic drugs in patients with schizophrenia who responded to the drug treatment.To test the relation of antipsychotic effect and occupancy more accurately, prospective study is needed. Nordström et al4.311C]NMSP had been used for the quantification of 5‐HT2A receptors.11C]MDL 10090718F]setoperon18F]altanserinMany second‐generation antipsychotics have the affinity for 5‐HT2A receptors in addition to the dopamine D2 receptors. Initially, DASB11C]MADAM[5.2One of the main mechanisms of antidepressant is the inhibitory effect to 5‐HTT. Meyer et al reported that clinical dose of four selective serotonin reuptake inhibitors (SSRI)  and venlafaxine showed over 80% 5‐HTT occupancy in patients with depression.5.318F]FMeNER‐D2, which showed suitable characteristic for the quantitative analysis of NET,11C]MeNER, which was previously developed with less optimal brain kinetics.Norepinephrine transporter (NET) inhibition is another mechanism of action of antidepressants. Compared to 5‐HTT, useful PET ligands for NET were not available when serotonin and norepinephrine reuptake inhibitors (SNRIs) were developed. DASB in phase I clinical study in Japan.Duloxetine is a SNRI , approved in Japan from 2010 , available in Japan from 2015. Meyer et al reported that 75 mg of venlafaxine ER showed over 80% 5‐HTT occupancy.Recently, the NET occupancy of venlafaxine ER in patients with depression was reported.5.5.3Milnacipran is an SNRI , approved firstly in Japan. Nogami et al reported that 25‐200 mg/d milnacipran showed 25%‐50% NET occupancy in patients with depression.5.5.4Nortriptyline is a classical antidepressant. It has relatively high selectivity to NET over 5‐HTT . Sekine et al reported that 75 mg of nortriptyline blocked 40% NET in healthy subjects.5.5.5Tramadol is an analgesic drug, which has opiate agonism as well as inhibitory effect for 5‐HTT and NET. Ogawa et al reported that 100 mg of tramadol showed 50% 5‐HTT occupancy in human brain using healthy subjects.5.5.6Quetiapine is approved as an antipsychotic drug, but anti‐depressive effect has been also reported. A possible explanation for anti‐depressive effect is that norquetiapine, a metabolite of quetiapine, has high affinity for NET. Nyberg et al reported that 300 mg of quetiapine XR blocked NET at 35% occupancy in healthy subjects.6As described above, carbon‐11 and fluorine‐18 radionuclides are used for the PET radioligands. Due to the short half‐life (around 20 minutes) of carbon‐11, the site production using a cyclotron and synthesis apparatus is required for the carbon‐11 PET radioligands. In case of fluorine‐18 PET radioligands , the delivery might be available. When multiple PET measurements in one day are considered to perform such as one baseline PET measurement and occupancy PET measurement with a drug, carbon‐11 PET radioligands are feasible due to the shorter half‐life.When the PET measurements are performed, radiation exposure from the radionuclides is needed to consider. Typical radiation dose from PET measurements is at similar level to that from computed tomography (CT) scans for the clinical usage or natural background radiation for a few years.Duration of PET measurement is relatively long, for example, 1 to 2 hours, and the subjects have to stay still during the measurement, which may make the participants feel uncomfortable especially in the patients with psychiatric/ neurodegenerative disorders. Simplified methods which can make the measurement time shorter should be considered if possible.7PET imaging has been widely used for the evaluation of CNS drugs. Especially, the estimations of dopamine D2 receptor occupancy for antipsychotics and 5‐HTT occupancy for antidepressants have played essential roles for dose finding in clinical trials. More evidence will be required to clarify the relationship between clinical effects and NET occupancy. Recently, the clinical targets for CNS drug development have been shifted to the negative symptoms/cognitive impairment of schizophrenia, treatment‐resistant depression, and so on. These clinical targets may involve glutamate, GABA, acetylcholine, and cannabinoid systems other than classical monoaminergic systems. PET imaging, together with concurrent development of adequate PET radioligands, would become more important technique for the evaluation of such new targets.AT is an employee of Takeda Pharmaceutical Company Limited. Other authors declare no conflict of interest.All authors had the substantial contributions to the conception of the work. RA wrote the initial draft of the manuscript. All authors revised it critically and approved the final version to be published.


EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for  Ewing sarcoma (EwS) is an undifferentiated small-round-cell sarcoma driven by FET-ETS fusion oncoproteins . EwS conIn more than 30% of cases, micrometastases are present at time of diagnosis . For theFrom a morphological point of view, histopathological diagnosis of EwS has been notoriously difficult due to its highly undifferentiated small-round-cell phenotype being shared with a variety of differential diagnoses , such asFUS, EWSR1, and potentially TAF15, with members of the ETS family of transcription factors  are largely pathognomonic for EwS [EWSR1-FLI1 and EWSR1-ERG, occurring in around 85% and 10% of cases, respectively.For almost three decades, the standard immunohistochemical marker for EwS has been the membranous glycoprotein CD99 ,26, whic for EwS ,33,34, m for EwS . The mosEWSR1 break-apart, can miss EwS cases with FUS-ETS fusions [EWSR1-ERG-positive EwS [EWSR1-WT1 fusions [EWSR1-NFATc2 fusion positive small-round-cell sarcomas [However, many current standard methods used for molecular diagnosis in pathology are not infallible. For instance, PCR analyses may fail to detect all FET-ETS variants unless multiplexed PCRs covering all known variants are employed ,36. In a fusions , are ofttive EwS , or can  fusions ,39,40 orsarcomas ,42.Therefore, a progressively increasing number of institutes nowadays employ more advanced approaches as auxiliary methods to confirm or rule out the diagnosis of EwS, such as next-generation sequencing (NGS) to directly sequence the fusion transcripts  and/or mBCL11B and GLG1, out of all 19,702 genes represented on the employed microarray platform, only ATP1A1 showed significant overexpression (log2 expression difference >2) in EwS compared to samples of the 20 morphological mimics tested [However, these sophisticated molecular diagnostic tools are not available at every institution worldwide and, in some cases, not feasible due to the quantity and/or quality of the available tumor specimen. Therefore, we strived to identify highly specific auxiliary diagnostic markers that can be detected by widely available and inexpensive immunohistochemistry (IHC) . Using cs tested . Hence, EWSR1-FLI1 target gene contributing to the malignant phenotype of EwS [GLG1 encodes a glycoprotein of still unknown function, even though it was first described in humans in 1990 [EWSR1-FLI1 to super-enhancer regions spanning their encoding genes [BCL11B is a transcription factor mainly involved in T cell development, which was previously identified as a direct e of EwS . GLG1 en in 1990 . In our  in 1990 , we founng genes . Althougng genes .EWSR1-ERG-positive EwS, and (III) determining the degree of intra-tumoral marker heterogeneity. To this end, TMAs of 453 cases including 133 EwS cases and 320 cases from 11 differential diagnoses were constructed and stained for BCL11B, GLG1, and CD99. Immunoreactivity was assessed independently by three researchers based on a modified Remmele and Stegner scoring system [In the current study, we aimed at (I) validating the high specificity of BCL11B and GLG1 overexpression for EwS in a new, much larger, and independent cohort, (II) testing the applicability of these markers also for g system .Our results confirm the high specificity of BCL11B and GLG1 for EwS regardless of the underlying fusion type, enabled the refinement of cut-off values for classifying cases as ‘positive’ or ‘negative’ for the given marker, and revealed intra-tumoral heterogeneity of the proposed auxiliary markers to be negligible in differential diagnoses for EwS. These findings lead us to propose a work-flow for diagnosing EwS on the basis of IHC detection of CD99, BCL11B, and GLG1, which could be widely applied independent of the availability of molecular pathology diagnostics.EWSR1 break-apart and immunohistochemical CD99 stains are commonly performed in pathological routine diagnostics for exclusion or diagnosis of EwS [EWSR1 break-apart occurs in several morphological mimics of EwS such as DSRCT harboring a pathognomonic EWSR1-WT1 fusion, and angiomatoid fibrous histiocytoma (AFH) commonly positive for EWSR1-ATF1 fusions  for detection of  fusions .CD99 is the standard IHC marker in pathological workup of potential EwS samples . Howevern = 133 molecularly confirmed EwS tumors and n = 320 morphological mimics from 11 differential diagnoses  cut-off of >0 to 88% for an IRS cut-off of >9 . HoweverEWSR1 break-apart in FISH in conjunction with immunoreactivity for CD99 are not fully sufficient to robustly diagnose EwS, suggesting that more sophisticated molecular pathology techniques, such as qRT-PCR, RNA-sequencing [Collectively, the representative cases shown in quencing ,52 or coquencing ,44, shouquencing . Indeed,To assess the diagnostic value of the proposed auxiliary markers BCL11B and GLG1, all 453 cases of our new cohort , were seWe observed that BCL11B and GLG1 were highly expressed in EwS. In fact, 75.9% of EwS cases (101/133) were highly reactive for at least one marker, co-expression of both was detected in 32.3% of EwS cases (43/133) a,b; 48.9As CD99 proved to be highly sensitive for EwS in our cohort, we considered only those samples as potential EwS with a CD99 IRS of at least 2  but moderately sensitive (64.7%) for EwS b. IntereEWSR1-FLI1), open slides of six molecularly confirmed EWSR1-ERG-positive EwS cases were stained for BCL11B and GLG1. Strikingly, BCL11B exhibited in all cases the highest possible IRS of 12 . In conjunction with the scoring results of the six molecularly confirmed EWSR1-ERG-positive EwS cases, these experimental results further suggest that high expression of both marker proteins in primary EwS is not restricted to EWSR1-FLI1-positive tumors.We previously showed that ion type . To testCIC-DUX4-positive sarcomas and 2 BCOR-rearranged sarcomas), which did not exhibit any BCL11B and GLG1 immunoreactivity. These findings in PDX models further support the high specificity of BCL11B and GLG1 for EwS.Since patient-derived xenografts (PDX) are increasingly used as preclinical models to study EwS biology , we testTumor material is often limited in pathological workup . In the context of histological tumor evaluation, limited material can lead to substantial confusion in case of high intra-tumoral heterogeneity of morphology and diagnostic marker expression. Therefore, the heterogeneity of scoring for CD99, BCL11B, and GLG1 in different cores per sample, originating from different regions of a given tumor block, was assessed. For non-EwS, 248 samples with three cores per marker were available in our TMA-cohort. Samples were classified as high or low for the different markers applying the thresholds defined above b.For CD99, 81% of non-EwS tumor samples were classified consistently across all three cores per sample, for BCL11B 97.6%, and for GLG1 and 94.8% , indicatn = 96 EwS cases two cores were present in each staining. In these 96 EwS cases, we found consistent classification as marker-positive or -negative in 81.1%–87.4%  and/or high GLG1 immunoreactivity . If both markers show immunoreactivity scores below the respective cut-off, the diagnosis of EwS should be molecularly confirmed a. ApplyiRobust diagnostics are essential to quickly and correctly assign EwS patients to the appropriate therapy. After initial clinical presentation and imaging, the diagnostic workup of potential EwS patients is performed by surgical pathologists. As EwS belongs to the group of undifferentiated small-round-cell sarcomas , its hisIn histopathological examination, evaluation of H&E-stained slides is routinely followed by CD99 immunostaining , which iEWSR1 gene [EWSR1 gene  [However, even strong membranous CD99 immunoreactivity is not specific for EwS ,55. IndeSR1 gene . Althougarcomas) ,40,41.FET-ETS fusion transcripts. However, due to the large variety of existing FET-ETS fusions in EwS [FET-ETS fusions. As a consequence, many institutions increasingly employ next-generation RNA-sequencing technologies to overcome these limitations. Yet, these techniques are not available or feasible in all cases, which is why a straight-forward immunohistochemical approach is still desirable.Another molecular technique is multiplex qRT-PCR to amplify pathognomonic s in EwS , many ofFET–non-FLI1 translocations [EWSR1-NFATc2-positive sarcomas [Caveolin-1, Cyclin-D1, NKX2-2, NR0B1 and PAX7 are known FET-ETS target genes [Hence, many groups have evaluated several potential diagnostic markers such as FLI1, NKX2-2, and PAX7 that could be used instead of the highly unspecific marker CD99 ,57,58. Hocations ,59, NKX2ocations  and can sarcomas ,57,58. Bsarcomas ,61, Cyclsarcomas , and NR0sarcomas  have beesarcomas ,64. Cavesarcomas  but its et genes ,65,66 imATP1A1, BCL11B, and GLG1 [To this end, we set out to identify highly specific auxiliary markers that can compensate the lack of specificity of CD99. By comparative transcriptome profiling of EwS cases and hundreds of samples from 20 morphological mimics, we previously identified three highly specific marker candidates, namely and GLG1 . Using Iand GLG1 . HoweverEWSR1-ERG fusions exhibited high marker immunoreactivity. In the current study, we confirmed the high specificity of BCL11B and GLG1 for EwS in a far larger and independent cohort of 133 EwS and 320 non-EwS from 11 differential diagnoses, including 118 samples from close morphological mimics such as DSRCT, neuroblastoma, and rhabdomyosarcoma. This high number of samples enabled a refinement of the IRS cut-offs for both markers . In conjunction with the highly sensitive marker CD99, our approach yielded a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC. Also, EwS cases with However, it should be noted that in our cohort, 36.8% of EwS cases could not be confirmed by this marker combination. If, in these cases, there is strong clinical suspicion of EwS, additional molecular approaches are mandatory for diagnosis. Additionally, in the current study, we used specific polyclonal antibodies against BCL11B and GLG1 (see methods). To further improve the accuracy of our diagnostic IHC approach and to reduce the risk of batch effects across different polyclonal antibodies, we recommend to systematically develop and improve monoclonal antibodies against both antigens that can be employed for IHC.2 tumor material). Potentially, the sensitivity might be even higher when more tumor material is available. As tumor material is often limited, heterogeneously expressed markers can lead to misdiagnoses depending on the tumor fraction captured in a given sample. Hence, homogeneous expression of potential markers within tumors is desirable. For non-EwS cases, the proposed marker combination of CD99, BCL11B, and GLG1 yielded a consistent classification over all 3 cores taken from different areas of each tumor sample in 95.2%. These findings suggest that the specificity of our approach is likely not compromised even when only little tumor material is available for diagnostics. Marker homogeneity in EwS was only moderate, possibly explaining the observed moderate sensitivity of our markers in our TMA cohort , especiThe study was conducted in accordance with the Declaration of Helsinki. Formalin-fixed paraffin-embedded (FFPE) human tissue samples of EwS and differential diagnoses were retrieved from the archives of the Institute of Pathology of the LMU Munich (years 2009 to 2019) and collaborating institutions with approval of the ethics committee of the LMU Munich University Hospital (307-16 UE). Patient derived xenografts (PDX) were provided by the Childhood Solid Tumor Network , D. Surdn = 453 samples is given in A total of 348 FFPE primary tissue samples and FFPE PDX samples from 64 pediatric sarcomas were collected. Only samples that could be diagnosed unambiguously using current state-of-the-art diagnostics for the respective tumor entity, were further processed. For each tumor, all FFPE blocks were examined in H&E stained slides and the tumor block containing the largest amount of vital tumor tissue was selected for TMA construction . Additionally, TMAs with 144 EwS samples and at least two cores per sample were obtained from W. Hartmann and U. Dirksen . The composition of the final cohort comprising n = 6 EWSR1-ERG positive EwS samples, n = 1 EWSR1-ATF1 positive angiomatoid fibrous histiocytoma (AFH), and n = 12 PDX models were used. Antigen retrieval was performed with microwave treatment, 2 × 15 min at 750 W using the antigen retrieval AR-10 solution  for GLG1 and the Dako Target Retrieval Solution  for BCL11B. After rinsing the slides with pH 7.5 TRIS buffer , endogenous peroxidase was blocked by 7.5% aqueous H2O2 solution at room temperature and blocking serum from the corresponding kits for 20 min.For IHC, newly cut 4 μm sections of TMAs or available open slides for Slides were then incubated for 60 min with primary rabbit-anti-human antibodies anti-BCL11B , or anti-GLG1 . Then slides were incubated with a secondary anti-rabbit IgG antibody  followed by target detection using AEC plus chromogen for 10 min .For IHC staining of CD99, 4 μm sections were incubated for 32 min with a monoclonal mouse-anti-human antibody  using the ultraView detection kit in a VENTANA BenchMark system .Immunoreactivity of all TMAs and open slides for CD99, BCL11B, and GLG1 was semi-quantitatively scored by three independent physician-scientists in analogy to scoring of hormone receptors with the immunoreactive score (IRS) established by Remmele and Stegner  with sliTo test whether the markers investigated in this study were heterogeneously expressed in a given differential diagnosis for EwS, we selected all tumor samples for which representative material was present in all three cores of the respective TMA for CD99, BCL11B, and GLG1 staining. In a next step, we evaluated for each core whether the IRS values for CD99, BCL11B, and GLG1 exceeded the predefined cut-offs  to classify each core as either marker high/positive or low/negative. Then, we assessed whether the results were consistent across all three cores per tumor sample. As most EwS cases were represented by two cores per sample, the evaluation of marker expression heterogeneity for EwS was performed separately on samples with two cores present in each staining.BCL11B and GLG1 by EWSR1-ERG, we employed clones of three EWSR1-ERG positive EwS cell lines  harboring a doxycycline inducible shRNA mediated EWSR1-ERG knockdown system. Wild-type CHLA25 and TC106 cells were obtained from the Children’s Oncology Group (COG), and EW3 cells were kindly provided by O. Delattre. Cell line authenticity was confirmed by STR-profiling and mycoplasma contamination was ruled out by nested PCR. Total RNA was extracted from these EwS cell lines 96 h after induction of EWSR1-ERG knockdown using the NucleoSpin RNA kit . RNA was reversely transcribed using the High-Capacity cDNA Reverse Transcription Kit . qRT-PCRs were performed with 1:10 diluted cDNA and 0.5 µM forward and reverse primers in SYBR Green PCR Master Mix (Applied Biosystems). Oligonucleotides were purchased from MWG Eurofins Genomics . The following primer sequences were used: BCL11B forward: 5′-ATGCCAGAATAGATGCCGG-3′;BCL11B reverse: 5′-CTCTATCTCCAGACCCTCGTC-3′;GLG1 forward: 5′-GTGGAGTGTAGAGATATAGTTGGC-3′;GLG1 reverse: 5′-ATCAGGTCCCCAGAGTCTATC-3′;RPLP0 forward: 5′-GAAACTCTGCATTCTCGCTTC-3′;RPLP0 reverse: 5′-GGTGTAATCCGTCTCCACAG-3′.To test for potential transcriptional regulation of RPLP0 was used as housekeeping gene [Reactions were run on a BioRad CFX Connect instrument  with the following thermal conditions: heat activation , DNA denaturation , annealing and elongation , 49 repeat cycles, final denaturation . ing gene .We anticipate that the here proposed diagnostic procedure a will pr


III(Cp*)(phen)Cl]+-Gr can exhibit redox and electrocatalytic properties for HER that are comparable to its homogeneous analogue via a two-electron reduction pathway. On the other hand, the one-electron reduction pathway is notably found to be less favorable kinetically and thermodynamically. Furthermore, molecular insights are provided with respect to the HER employing molecular orbitals analyses and mechanistic aspects. Importantly, our findings may provide insights toward designing more efficient graphene-based molecular heterogeneous electrocatalysts for more efficient energy production.The heterogeneous metal-based molecular electrocatalyst can typically exhibit attractive features compared to its homogeneous analogue including recoverability and durability. As such, it is necessary to evaluate the electrocatalytic behavior of heterogenized molecular catalysts of interest toward gaining insights concerning the retainability of such behaviors while benefiting from heterogenization. In this work, we examined computationally the electrochemical properties of nanographene-based heterogenized molecular complexes of Rhodium. We assessed, as well, the electrocatalytic behavior of the heterogenized molecular catalyst for hydrogen evolution reaction (HER). Two electrochemical pathways were examined, namely one- and two-electron electrochemical reduction pathways. Interestingly, it is computationally demonstrated that [Rh In principle, these electrocatalysts are generally synthetic in nature that comprise transition metals coordinated with a variety of ligands. Commonly, these electrocatalysts are synthetically prepared and tested as homogeneous molecular electrocatalysts for various types of important chemical transformations of interest, such as the hydrogen evolution reaction (HER)10. Prospectively, the heterogeneous versions of such electrocatalyst can be advantageous in terms of robusticity and practicality toward their potential integration within devices for redox-mediated energy conversion. Hence, great interests have recently grown toward such heterogenization of molecular electrocatalysts using various types of nanomaterial, such as carbon nanotubes and graphene14.Molecular electrocatalysts have recently gained great level of interests due to its practicality in promoting various chemical transformations via redox mediation for energy applications17. However, for the heterogenized molecular electrocatalysts, it must be mentioned that efficient electronic coupling between the electrode and molecular catalysts is crucial toward retaining the characteristics electrocatalytic properties of the homogeneous analogue while functioning as heterogeneous electrocatalysts. Notably, recent approaches have rationalized the covalent attachment of the molecular homogeneous catalysts to the surface of electrochemically active carbon materials. For example, Surendranath et al. have reported on the immobilized heterogeneous catalyst of Rh metal complexes on graphite-conjugated catalyst linked via pyrazine groups18. In their work, they demonstrated experimentally that the homogeneous molecular catalysts [RhIII(cp*)(bpy)Cl]1+ can exhibit different redox behavior compared to the heterogenized analogue in acetonitrile as they undergo two-electron metal-centered and one-electron non-metal-centered reduction pathways, respectively. However, although many studies have been reported since the early work of Grätzel and Kölle concerning the catalytic properties of the homogeneous analogues of Rh complexes26, no results have been yet reported concerning the assessments of the catalytic behaviors of the heterogenized molecular catalysts for the HER.Efforts concerning heterogenization of molecular electrocatalysts have focused on the attachment of the homogeneous molecular catalysts to the surface of nanomaterials serving as electrodes for redox mediation catalysis of reactions of interests. Various approaches have been developed in this regards, this includes self-assembled monolayers (SAMs), or via noncovalent immobilization such as π-π stacking4-Cp*H] as a ligand29.Importantly, as mentioned above, the cruciality of efficient electronic coupling to the electrode necessitates evaluating the redox behavior of such molecular electrocatalysts before and after heterogenization and consequently evaluating their catalytic behavior toward a chemical transformation of interest, such as the HER. On the other hand, the applicability of Rh(Cp*)-based homogeneous molecular catalysts in acetonitrile has recently been interestingly addressed toward electrochemical hydrogen generation. The corresponding catalytic cycle Fig.  comprise34. In the light of the importance of heterogenization of transition metal-based molecular electrocatalysts and inspired by the recent work of Surendranath et al., we aimed in this work using Density Functional Theory (DFT) to computationally evaluate the redox behavior of selected graphene-based heterogenized molecular Rh complexes ([RhIII(Cp*)(L)Cl]1+) and the corresponding electrocatalytic activity for the HER. Both behaviors of interest, namely redox and electrocatalysis of the HER, are comparatively evaluated for both phases at the molecular levels. The assessment includes selectively redox active species as displayed in Fig. Importantly, there is a growing necessity toward providing computational-based molecular interpretations and insights concerning the electrocatalytic properties of the molecular heterogeneous electrocatalysts compared to its homogeneous analogue, which in turn is significant for improving their performance towards developing electrocatalyst by design35. Geometry optimization of all complexes were performed using DFT method with B3LYP functional followed by frequency calculations. 6–31 G(d) basis set was used for the non-metals, C, H, N, and Cl atoms, whereas lanl2dz for Rh. The graphene sheet (Gr) was modelled as four fused benzene. Selected pentamethylcyclopentadiene-based Rhodium complexes [RhIII(Cp*)] are considered in this work. Figure III(Cp*)(L)Cl]1+  (bpy) or phenolphthalein (phen). For the calculations of the solvent effects, an implicit solvation method of the integral equation formalism variant of the Polarizable Continuum Model (IEFPCM) was used in acetonitrile (ε = 35.688).All calculations were conducted using Gaussian09 software package, Revision E.0137. The change in standard Gibbs free energy of the reduction of the corresponding substance in solution is ) and the corresponding o (Fc+/0))38, were calculated using Eqs. The mentclass2pt{minim+ (L= bpy or phen), such complexes commonly undergo a metal-centered two-electron reduction (RhIII/I) at approximately −1.19 V vs Fc+/0,with simultaneous dissociation of chloride ion (Cl−)22; whereas the heterogenized analogue undergoes a nonmetal-centered one-electron reduction at −1.29 V vs Fc+/0 with alike dissociation of Cl18. As such, we optimized the geometries of these species that are involved in the electrochemical reduction using DFT calculations. Coordinates of the optimized geometries in acetonitrile are provided in the supplementary information.The analyses of interest here are focused on insighting the redox and the corresponding electrocatalytic behaviors of the metal-based heterogenized molecular catalysts of selected Rhodium complexes compared to the homogenous analogue in terms of structural properties, mechanistic aspects, and molecular orbitals. It must be mentioned that performing such evaluating protocols requires initially performing accurate DFT-based geometry optimizations for all species that are involved in the electrochemical reduction and catalytic cycle of interest. According to previously reported experimental results concerning the redox behavior of complexes of [Rh(Cp*)(L)Cl]−. Notably, such breakage of the Rh-Cl bond was obtained for both processes of two-electron and one-electron electrochemical reduction. On the other hand, the DFT based calculated geometrical properties were benchmarked with the reported XRD crystallographic data by Blakemore et al. for analogous complexes; particularly, [RhIII(Cp*)(bpy)X]+ 40. We emphasized herein on the geometrical properties after the electrochemical reduction process. It must be mentioned that the experimental data was reported for the Bromide ligand, whereas the calculation here was performed for the Chloride ligand. However, in both cases, the product of the electrochemical reduction must have the same structure as of ejecting the halogen ion ligand. For example, bond lengths for Rh-C(Cp*) in the range of 2.211–2.253 Å was obtained in vacuum and acetonitrile for [RhIII(Cp*)(bpy)]2+ after the two-electron reduction process, which are in good agreement with the corresponding experimental values of 2.190–2.223 Å. For the heterogeneous analogues, Rh-C(Cp*) in the ranges 2.239–2.295 and 2.264–2.330 Å, were obtained after the one-electron and two-electron reductions processes in vacuum, respectively, indicative of ignorable change in the corresponding bond length after heterogenization and independent of the number of electrons transferred. Furthermore, similar behaviors were obtained for the C-C bonds length within the Cp* and bpy ligands. Furthermore, a notable change in the geometry of the reduced forms in both phases is the dihedral angle between the plane of phen containing Rh and the Cp* ring; see Fig. III(Cp*)(bpy)]2+ after electrochemical reduction40.Figure o of solvation needed for calculating the redox potential in the corresponding medium (infra vide). Indeed, the DFT calculations revealed a negligible effect of solvation (acetonitrile) on the structural properties of the examined systems, this includes the main bonds length and angles of atoms that can be potentially involved directly in the catalytic and electrochemical behavior of the molecule.Importantly, it is noteworthy to mention that this electrochemically induced dramatic change in the geometrical properties of the electrocatalyst might be crucial to initiate the binding to substrate of interest that in turn to be catalytically converted, such as the HER. In addition, as mentioned in the methodology section, performing the DFT calculations in different solvents is essential for obtaining the ∆G2. As such, the thermodynamics quantities obtained from the DFT calculations, single-point frequency calculation of the optimized geometry in particular, were utilized for calculating the change in free energy of each step illustrated in Fig. 37. As some functionals perform very well for such prediction for some substances, they totally fail for others based on benchmarking against experimental values. For example, as recently reported by Liang et al.36, B3LYP functional with a combination of LANL2DZ and 6–31 G(d) basis set for metal and non-metal atoms, respectively, and IEFPCM implicit solvation model, perform quite-well for wide range of organometallic substances with mean absolute deviation (MAD) of 0.233 V; yet, this method fails for some other substances. Nevertheless, considering the main two factors in computational chemistry, namely accuracy and computational cost, this method was highly recommended36. To this end, the +/0) of the molecular reduction models of two-electron and one-electron for the homogeneous and heterogenized analogue, respectively. However, further calculation were conducted for the presumable 2-electron reduction of the heterogenized electrocatalyst. Importantly, the validity of the calculation method applied in this work is evaluated through benchmarking with reported experimental values wherever applicable. As such, these theoretical values were calculated versus the appropriate reference electrode as indicated by the reported experimental results, namely Fc+/0 36.It is noteworthy mentioning that the electrochemical assessment of a potential electrocatalysts is crucial toward developing efficient electrocatalysts of interest of relatively low overpotential, which in turn is highly desired for electrocatalytic production of H36. However, a notable discrepancy for the calculated III] can generally undergo two-electron electrochemical reduction that is observed experimentally as a single process41. Further attempts were conducted to reduce the discrepancy between the calculated and the experimental value of the 1-electron reduction process. In this regard, we increased the number of the benzene fused rings to seven and we examined the effect of another DFT functionals, namely M06; see Fig. +/0) calculated for the one-electron reduction of A-Gr. Adding more fused rings has shown no improvement on the +/−. As noted in Table +/− afforded a +/0 that is more comparable with the experimental values of the homogeneous analogue of various members of the [Cp*RhIII] family.It is noteworthy to mention that the key point herein is to examine the effect of heterogenization on the redox potential of the Rh molecular electrocatalyst. All results are compiled in Table mentclass2pt{minim22. For selected Cp*Rh complexes, Henke et al. reported +/0 in acetonitrile) of −1.25 V for the 4 and 4′ t-butyl disubstituted phen ligand compared with −1.21 V for the unsubstituted one. However, notable decrease in the 3 substituents at the same positions of substitution. Nevertheless, it is highly anticipated that extending the range of such substituents may help in tuning the n-electron reduction (n:1 ↔ 2) of the heterogenized analogue of the molecular electrocatalysts24.These DFT results indicate that the electrochemical molecular properties of the species considered in this study are slightly dependent on the types of the diamine ligands, namely phen and bpy. This, in turn in good agreement with the recently reported results for such effects4-Cp*H] is the main intermediate that is formed during the catalytic cycle of HER; yet, other intermediates can still be potentially formed including the metal-hydride species42. In the work of Johnson et al.29, they examined using DFT with M06 functionals the mechanism of HER using homogeneous molecular catalysts of Cp*Rh(bpy). Still, in our work we utilized different functional for studying the process of interest the decisive approach to be followed is based on the benchmarked analysis of the calculated reduction potential of the molecules of interest. Indeed, the approach that we utilized afforded relatively better performance concerning the electrochemical properties, and hence, for the purpose of consistency, we employed the same approach for investigating the underlying mechanism of the HER suing the homogeneous molecular catalysts of Cp*Rh(bpy) and their graphene-heterogenized analogues. Decisively, as the primary focus of this work is to consider a comparative approach concerning the homogeneous molecular catalyst of Rh versus its heterogenized analogue toward the catalyzed HER, we focused on examining the non-innocent role of [Rh-η5-Cp*] ligand and the corresponding transformation to its protonated analogue [Rh-η4-Cp*H] as the key transformational step within the catalytic cycle of the HER that is in turn essential for initiating the process. Hence, the objective of this approach is to provide quantitative thermodynamic and kinetic criteria toward comparative evaluation of a heterogenized molecular catalyst versus its corresponding analogue. On the other hand, protonation of the Cp* ligand was modeled explicitly using a weak acid, namely Et3NH+. As such, the energy diagram obtained by free energies differences for such catalytic cycle comprising the species of interest is displayed in Fig. Importantly, the significance of heterogenization of a molecular electrocatalyst can be generally assessed through investigating their catalytic performance toward a reaction of interest, such as the HER; this includes evaluating the role of n-electron reduction. As illustrated in Fig. e of Rh-η-Cp* liga# and ∆G° were assessed, which refer to the thermodynamics related to the formation of the excited state and the products, respectively. As it can be noticed from the energy profile, A1 and A2 exhibited approximately an identical catalytic behavior kinetically and thermodynamically with a negligible difference in ∆G# and ∆G° of only 0.8 and 0.9 kcal/mol, respectively. Likewise for the two-electron reduction pathway of A-Gr+/− compared to the homogeneous analogue of A1 and A2, where almost the same typical behavior is repeated. For example, the DFT calculations afforded ∆G# and ∆G° of −29.4 and −18.6 kcal/mol, respectively, indicating a feasible kinetic barrier and spontaneous process for forming the protonated ligand complex [η4-Cp*H], which in turn facilitating expectedly the imitation process of the HER. However, as revealed by the DFT calculations, it is evident that the formation of [RhI (η4-Cp*H)]-Gr through a two-electron reduction process is kinetically and thermodynamically favored over the one-electron reduction process to form [RhII (η4-Cp*H)]. Other important factor to be mentioned herein is the solvent effect. Notably, the implicit solvation effect has increased the activation energy of the protonation step from 7.2 kcal/ mol in vacuum for A-Gr+/− into 29.4 kcal/mol in acetonitrile. Alike solvation behavior was observed for all examined species. Furthermore, another issue that is worth to be addressed structurally is the bonds’ length of atoms that are most likely to be involved in the catalytic process of the HER. A1, A2, and A-Gr+/− exhibited similar structural properties including those of the main bonds involving Rh. For example, for the protonated complexes of A, A2, and A-Gr+/− in acetonitrile, almost identical bond length of 2.160 ) and 2.680 Å were calculated for Rh- CCp* and Rh- CCp*(H), respectively. However, although a bond length of 2.80 Å was calculated for Rh- CCp*(H) of A-Gr+/0, two distinctive bonds length was observed for the other four carbon atoms of (η4-Cp*H) with an average of 2.374 and 2.253 Å.In Fig. 2, where a lower energy for the LUMO can desirably assure lower electrochemical overpotential without significantly affecting the overall catalytic efficiency. As such, we conducted a simulation for the MOs of interest including the frontier ones for all complexes considered in this study. Hence, one approach of analyses is visualizing the MOs of the heterogenized molecular catalyst and compare it with the MOs of its corresponding homogeneous analogue. Selected MOs of all [Rh(Cp*)(phen)Cl]1+ before and after heterogenization are displayed in Fig. To provide further insights at the molecular level concerning the catalytic behaviors toward the HER of the species considered in this study, we performed molecular orbitals analyses. It is noteworthy mentioning that providing insights regarding the properties of the frontier molecular orbitals, namely highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), of a catalysts is important toward enhancing the understanding of the corresponding electrochemical catalytic behavior. In principle, the latter is significantly important in electrochemical catalyzed reduction of substances of interest, such as H+/−, one-electron reduction A-Gr+/0, and two-electron reduction A-Gr+/− are displayed in Fig. −, it is expected that the additional two electrons of reduction will be placed in the LUMO to afford new HOMO with calculated stabilization of 0.0404 eV from −0.0979 to −0.1383 eV. This new HOMO as can be noticed in Fig. 2 metal orbital with no interaction with the surrounded ligands with an energy of −0.1937eV. However, in correspondence with the metal centered two-electron reduction process, it must be mentioned that such process may have shifted the relative energy of the MOs to place the dz2 metal orbital of Rh in the position of HOMO-1 as revealed by the DFT calculations. Comparing this behavior with the heterogenized analogue, it can be noticed that after heterogenization the positions of the LUMO and HOMO have changed with the addition of new MO from the conjugated system of the pyrazine linker and graphene. The LUMO has shifted to LUMO+1 with an energy of −0.98 eV and HOMO has shifted to HOMO-2 with an energy of −0.244 eV, which indeed is more stable compared to the HOMO of the homogenous analogue. Nevertheless, the two-electron reduction has interestingly afforded a new HOMO of the same shape of comparable energy as of the homogeneous analogue. This new HOMO in fact is produced from the LUMO+1 rather than the LUMO. Importantly, this new HOMO, regardless of its source, may expectedly processed with the same catalytic behavior for the HER, which in turn is consistent with the similarities in the It is indeed important to consider the localization pattern and energies of the MOs and their corresponding changes during the catalytic reaction for both transformations, namely before and after heterogenization, as well as the influence of electrochemical reduction. Selected MOs of A1− were analyzed. It is noteworthy to mention that the addition of Cl− will relatively affect the energy of the MOs, and hence the comparison herein is based on qualitative aspects. Also, the MOs that are considered herein are the occupied ones that may exhibit major contribution in the catalyzed HER. Selected occupied MOs of A1+/−, one-electron reduction A-Gr+/0, two-electron reduction A-Gr+/− after protonation are displayed in Fig. Furthermore, The MOs before and after protonation accompanied by the re-coordination with the Cl+ to form the protonated species. For the protonated species of the homogeneous catalysts and its heterogenized analogue (2e), and considering the re-coordination of Rh with Cl−, a new HOMO is formed that is localized on the metal and chloride ligand indicating that further protonation will most likely be a metallic-centered process, which in turn can be consequently followed by evolution of H2 and regenerating the original catalyst. However, the protonated 1e specie of the heterogenized catalyst (A(1e)-Gr-H+) exhibited a different behavior, where the SOMO is localized on the graphene ligand. This new SOMO in turn may shield the SOMO-1 orbital, which is of the same characters as that of the HOMO of the A(2e)-Gr-H+, and hence may retard further processes for the HER. In fact, this non-metallic centered SOMO is more likely consistent with the non-metallic electrochemical one-electron reduction of the heterogenized catalyst that was observed experimentally18.As shown in Fig. III(Cp*)(phen)Cl]+ can exhibit typical two-electron redox behavior and electrocatalytic properties for the HER that are comparable to its behavior as homogeneous electrocatalyst. Importantly, the appropriateness of the DFT-based redox calculations employing Born–Haber cycle in combination with IEFPCM solvation model in acetonitrile is benchmarked through comparison with alike experimental systems that were previously reported. The appropriate modelling of nanographene sheet was simulated and successfully demonstrated through implementation of a four-fused rings, where it is demonstrated that adding more rings to the model did not enhance the accuracy of the calculations. Furthermore, it is demonstrated through the DFT calculations that the two-electron reduction pathway can afford more favorable kinetic and thermodynamic pathway for the HER compared with the one-electron pathway, which in turn is in good correspondence with the behavior of the homogeneous analogue. However, with the high value obtained for the one-electron reduction potential of the heterogeneous catalysts, it sounds challenging to utilize such graphene-based covalently heterogenized molecular catalysts of Cp*Rh for experimental H2 evolution. Interestingly, the findings reported herein may aspiringly be used for further designing of novel and more efficient heterogeneous molecular catalysts.We have demonstrated computationally using DFT approach that the nanographene-pyrazines-based covalently heterogenized molecular catalysts of [RhSupplementary Information.


In vitro viability tests using the incubation of 3T3 fibroblast cells in a nanofiber-based matrix did not reveal any adverse effects, confirming the biocompatibility of hybrid nanofiber structures. The generated hybrid nanofibers functionalized with plasmonic TiN NPs are promising for the development of smart scaffold for tissue engineering platforms and open up new avenues for theranostic applications. Herein, we report the fabrication and characterization of novel polycaprolactone (PCL)-based nanofibers functionalized with bare (ligand-free) titanium nitride (TiN) nanoparticles (NPs) for tissue engineering applications. Nanofibers were prepared by a newly developed protocol based on the electrospinning of PCL solutions together with TiN NPs synthesized by femtosecond laser ablation in acetone. The generated hybrid nanofibers were characterised using spectroscopy, microscopy, and thermal analysis techniques. As shown by scanning electron microscopy measurements, the fabricated electrospun nanofibers had uniform morphology, while their diameter varied between 0.403 ± 0.230 µm and 1.1 ± 0.15 µm by optimising electrospinning solutions and parameters. Thermal analysis measurements demonstrated that the inclusion of TiN NPs in nanofibers led to slight variation in mass degradation initiation and phase change behaviour (T The development of novel platforms for regenerative medicine forms one of the attractive applications of nanotechnologies and newly synthesized inorganic nanomaterials, which can be used in combination with organic matrices for tissue engineering ,2,3. HerVarious polymers with varying molecular weights, biodegradability and hydrophilicity have been successfully electrospun in the form of homogenous nanofiber structures. Such nanofibers can have unique properties such as high surface area and pore size, but their functional domain remains limited. To enable new functionalities, one can decorate the nanofibers with functional agents such as inorganic nanoparticles (NPs) or biomolecules ,15,16. A3+ is typically required to stabilize the nanofibers and adapt them for utilization in biological systems. Pulsed laser ablation presents a “physical” alternative to fabricated nanomaterials, which are free from stabilizing agents and impurities ,23. Thisin vitro and in vivo, as well as initiating a strong photothermal therapeutic effect under near-infrared laser irradiation in the region of relative tissue transparency. It is also important that PCL is soluble in acetone, while femtosecond laser ablation makes possible the synthesis of stable solutions of TiN NPs in acetone, which simplifies their co-electrospinning.We believe that the properties of nanoparticle-decorated hybrid nanofiber matrices can be further improved by the choice of appropriate materials as building blocks for such structures. Here, polycaprolactone (PCL) looks to be a promising polymer for the formation of nanofiber matrices due to the combination of biocompatibility and biodegradability options ,38,39, aIn this work, we report on the successful electrospinning of PCL together with laser-synthesized TiN NPs at various ratios in order to fabricate a novel hierarchical hybrid nanofiber platform for tissue engineering. The obtained nanofibers were characterized by panel materials analysis techniques such as scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and thermal analysis (TGA and DSC). Nanofibers containing various concentrations of TiN NPs were further studied to compare their influence on cell growth behaviour and the overall cytotoxicity of functionalized nanofibers.w = 80 kD) from Purasorb®, Corbion, Netherlands. Dichloromethane (DCM), acetone  and ethanol from Carl Roth, Karlsruhe, Germany were used as solvents. A TiN (99.99%) pellet from GoodFellow, Cambridge, United Kingdom was used as the target for the synthesis of TiN NPs. Commercial Polycaprolactone (PCL) powder  laser ablation of bulk TiN target in acetone, as was earlier described in . Brieflyw/v) in DCM (3 mL) and acetone/ethanol (2 mL). A 5 mL solution was prepared for each concentration for electrospinning, as described in w/v) PCL was chosen as the optimised concentration for functionalization with TiN NPs. Solutions of the polymer with TiN NPs at various concentrations were solubilized in DCM and acetone in the ratio of 3:2 (v/v).Electrospinning was performed using 7 different solutions, increasing the concentration of PCL until homogenous fibers were not identified in SEM micrographs. Two solutions were prepared using PCL, starting with the concentration of 8% , which were inserted into a spinneret. Syringes were placed on a programmable pump to control the flow rate during electrospinning. The flow rate for all solutions was fixed at 0.2 or 0.3 mL hTM, Freising, Germany) was used at the accelerating voltage of 20 kV. The observation was carried out after scratching the fibers from the aluminium foil and fixing them onto the stub using carbon tape, which was attached to the stage. A DSM 982 Gemini Zeiss system  at the accelerating voltage of 20 kV was used to provide higher magnification micrographs after the functionalization process. ImageJ® and OriginLab software were used for analysis and graphical representation of the obtained nanofibers images.To characterize the size and surface morphology of electrospun nanofibers, a scanning electron microscope  . For each sample, 7.5 mg of scratched nanofibers from aluminium foil were placed in a ceramic cuvette at a nitrogen flow rate of 20 mL min−1. The samples were heated at 10 °C min−1 from 30 °C to 700 °C.Differential scanning calorimetry (DSC)  was used to characterize the thermal properties of formed nanofibers. The samples were subjected to multiple heating cycles at a rate of 10 °C minA FTIR/ATR technique was applied to nanofibers and polymers in their solid state to distinguish the specific vibrational frequency of pristine nanofibers and its changes due to functionalization. FTIR/ATR spectra were recorded using a PerkinElmer Spectrum 2000 spectrometer .We performed the basic cytotoxicity tests that are commonly used in in vitro studies, including metabolic activity, cell proliferation, cell adhesion and live/dead staining assays. We used mouse 3T3 fibroblasts, clone A31, which are used as a standard cell line for cytotoxicity testing.3 cells cm−2 and cultured in Dulbecco´s Modified Eagle´s Medium , supplemented with 10% fetal bovine serum , 100 I.U. mL−1 penicillin, and 100 ug mL−1 streptomycin . Four samples were seeded for each item for MTS/DNA testing, three samples for the live/dead test and three samples for DiOC6(3)/propidium iodide staining.Nanofibers were cut to circles with a diameter of 6 mm and sterilized in 70% ethanol for 30 min, washed twice with phosphate-buffered saline (PBS) and seeded with mouse 3T3 fibroblasts at a density of 25.8 × 10® AQueous One Solution Cell Proliferation Assay  after 1, 3, 7, 10 and 15 days. Scaffolds were put into new wells with 100 μL medium and 20 μL MTS solution and incubated in a CO2 incubator for 2 h. Then, absorbance of 100 μL of solution was measured using an Infinite M200 Pro spectrophotometer . Scaffolds without cells were used as negative controls.Metabolic activity was evaluated using CellTiter 96DNA amount was measured using a Quant-iT tm dsDNA assay . Fluorescence was measured using a spectrophotometer  and dsDNA amount was counted from the calibration curve of standards. −1 DiOC6(3) staining  was used for cell visualization on day 1. After 45 min of incubation with DiOC6(3), cells were incubated with 5 μg mL−1 of propidium iodide for 8 min, washed with PBS and visualized using a confocal Zeiss LSM 880 Airyscan microscope at λexc = 488 nm and λem = 505–515 nm for DiOC6(3) and λexc = 561 nm and λem > 576 nm for propidium iodide.1 μg mL−1 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester , which was dissolved in serum-free medium and incubated with the scaffolds for 45 min. The scaffolds were then incubated with 5 μg mL−1 of propidium iodide (in PBS) for 8 min. Cells were visualized using a confocal microscope, Olympus FV10i, at λexc = 488 nm and λem = 505–515 nm for BCECF-AM (living cells) and λexc = 561 nm and λem > 576 nm for propidium iodide (dead cells).Live/dead staining was performed using 1 μg mLStatistical analysis was performed using SigmaStat 3.5 software . One-way ANOVA and the Student–Newman–Keuls method were used for data analysis. The level of significance was set to 0.05.w/v) concentration was selected as the optimal one. This concentration was later used in the co-electrospinning of PCL and TiN NPs.Several nanofiber formulations based on PCL solutions were prepared by electrospinning and then examined using SEM to optimize the parameters of nanofibers before their functionalization with TiN NPs. Then, the nanofibers were fabricated using various concentrations of TiN NPs. ® software [Based on the optimized parameters of PCL solutions, we then performed electrospinning of PCL together with TiN NPs at different concentrations. Typical SEM images of hybrid nanofibers prepared under different nanoparticle concentrations are shown in software  was usedsoftware .The presence of TiN NPs on the surface of PCL nanofibers can be clearly identified in SEM images, as shown in Thermal analysis was carried out using TGA. Our tests did not reveal any major difference in maximal degradation temperatures and degradation initiation temperatures when pristine nanofibers and nanofibers functionalized with TiN NPs were used. All samples followed an identical trend with a single-step degradation and similar degrading temperatures. The degradation initiation temperature was calculated with a minimum of 5% mass loss for the samples . Pristin−1), started to degrade at a lower temperature of about 100 °C, which could be attributed to an insufficient number of NPs within the fibers matrix to dissipate heat. The increase in concentration of TiN NPs improved the stability of nanofibers at low temperatures due to the increase of heat capacity. Here, sample T20_0N6 with the highest concentration of TiN NPs (2 mL at 0.45 mg L−1) demonstrated similar degradation behaviour to pristine nanofibers. It should be also noted that most of the organic part degraded at the end of the program cycle. Indeed, about 0.1% of the initial mass was available at the cycle’s end for pristine nanofibers, while the relevant values for functionalized nanofibers were in the range of 0.2–0.35%.Sample T20_0N2, prepared under a relatively low concentration of TiN NPs (2 mL at 0.15 mg mLAs one can see from derivative thermogram (DTG) curve of the hybridized nanofibers , the prem) for pristine nanofibers and sample T20_1N1 was observed at 57.5 and 59 °C, respectively. The area under the peak showing enthalpy change (ΔH) was measured at 9.5 mWmg−1 for pristine nanofibers, while nanofibers functionalized at the lowest concentration of TiN NPs (T20_1N1) demonstrated a very slight increase in enthalpy up to 11.4 mWmg−1  tests were then undertaken to further examine the changes in thermal behaviour of functionalized nanofibers. The samples were analysed during two heating cycles, but we did not reveal differences between data from these cycles. DSC data from the first heating cycle are shown in 4 mWmg−1 . Nanofib−1, which is consistent with the literature [−1, while a weak peak at 1600 cm−1, related to acetone, is resolvable in the case of the pure nanoparticle solution. C-O stretched bonds from 1200 to 1160 cm−1 are also resolvable for all samples containing PCL [Functionalized and non-functionalized nanofibers were then studied using FTIR/ATR spectroscopy to analyse changes in infrared absorption peaks. Results of FTIR/ATR examination are displayed in terature ,45. One ning PCL . It is aIn MTS assays, absorbance increased every experimental day from day 1 until day 15 for all scaffolds. As shown in As shown in Inorganic nanomaterials have already been explored in various clinical applications. As an example, silver NPs were found to improve wound healing in both acute burn wounds and chronic wounds , while z2 nanofibers and TiO2 NPs in a dose range of 2.5–80 μg cm−2 were tested for the viability of Raw 264.7 (macrophages) and adenocarcinoma epithelial cells A549 [2 nanofibers and NPs induced low production of ROS species. TiO2 nanofibers changed the normal round morphology of macrophages to spindle shaped [2 nanospheres of longer fibre structures over 15 μm initiated an inflammatory response via alveolar macrophages and the production of inflammatory cytokines similar to silica or asbestos [TiOlls A549 . At the e shaped . On the asbestos .x) in thin layers prepared by atomic layer deposition on cellulose fibers demonstrated the layer depth-depending adhesion of human adipose-derived adult stem cells, with the highest adhesion occurring for samples with 20 Å coating [Owing to high wear resistance, high hardness, high scratch resistance, a low friction coefficient and high wettability, TiN-based materials have a wide spectrum of applications as coatings for articulating surfaces of implants. This material is approved by FDA and considered as physiologically inert and non-carcinogenic. Prolonged exposure of biological objects and tissues to TiN does not cause any toxic effects . Titaniu coating . −1 solution) [Polished discs of pure titanium ASTM F67, titanium alloy (Ti-6Al-4V) ASTM F136 (10), stainless steel ASTM F138, and cobalt alloy (Co-Cr-Mo) ASTM F75 were explored through corrosion tests in conditions of bare and TiN layer-coated surfaces. 2.2 μm- height TiN coating of stainless steel increased its corrosion resistance, but no positive effect was seen on pure Ti or Ti-6Al-4V. Interestingly, the TiN coatings of these materials exhibited no cytotoxicity, intradermal irritation, or acute systemic toxicity response  . In our experiments, TiN NPs were incorporated in PCL, a biocompatible polymer, which excludes the direct exposition of cells to potentially toxic TiN NPs. The preliminary results of the biological tests showed good biocompatibility of all tested materials, with increased metabolic activity on day 10 and the highest increase of dsDNA content on day 15. Here, T20_0N6 samples with the highest concentration of TiN NPs (4 mL) demonstrated the best metabolic activity. However, in both TiN NPs samples and PCL, some areas with dead cells were observed on day 1 and 15. As the presented in vitro tests did not show fully biocompatible material, further long-term in vitro tests over at least 5 weeks must be performed. It is important that the fabricated hybrid organic–inorganic nanofiber structures can combine good biocompatibility of PCL and specific physical properties of inorganic TiN NPs. In particular, the plasmonic and catalytic properties of TiN NPs can be used, e.g., for the phototherapy of cancer and its combination with chemotherapy and radiotherapy, which are especially effective during the G2 phase of the cell cycle and can be enhanced by optimizations of pH, microcirculation, metabolism, and the presence of sensitizers .We have demonstrated a simple methodology to fabricate functional hybrid nanofibers based on the co-electrospinning of polycaprolactone solutions and bare laser-synthesized TiN NPs. The parameters of electrospinning using 80 kD molecular weight PCL were optimized to obtain the desired morphology and properties of the nanofibers. It was found that with 20% (w/v) concentration of PCL dissolved in dichloromethane and acetone at a 3:2 volume ratio, the fabricated nanofibers had optimal parameters for further functionalization with TiN NPs. We then fabricated hybrid nanofibers by electrospinning PCL with TiN NPs at different concentrations. Statistical analysis showed that the diameter of nanofibers became more uniform when PCL was electrospun with TiN NPs, while the presence of TiN NPs in the system was confirmed by high-resolution scanning electron microscopy, where NPs could be observed being attached to the polymer matrix or partially embedded. The examination of fiber nanofibers by DSC and TGA analysis revealed that TiN NPs provided slightly different characteristics compared with pristine nanofibers, which could be attributed to better thermal distribution within the hybrid matrix. The potential of hybrid nanofibers in biomedical applications was further assessed by biological assessment, which showed that NPs-decorated nanofibers demonstrated good biocompatibility in vitro, identical to pristine PCL nanofiber samples. The obtained results evidence that novel hybrid matrices based on PCL nanofibers and TiN NPs can serve as excellent candidates for tissue engineering, drug delivery agents and cancer theranostics.


Caste polymorphism in eusocial insects is based on morphological plasticity and linked to physiological and behavioral characteristics. To test the possibility that dopamine production in the brain is associated with the caste-specific morphology and behavior in female honey bees, an intermediate caste was produced via artificial rearing using different amounts of diet, before quantifying the dopamine levels and conducting behavioral tests. In field colonies, individual traits such as mandibular shape, number of ovarioles, diameter of spermatheca, and dopamine levels in the brain differed significantly between workers and queens. Females given 1.5 times the amount of artificial diet that control worker receives during the larval stage in the laboratory had characteristics intermediate between castes. The dopamine levels in the brain were positively correlated with the mandibular shape indexes, number of ovarioles, and spermatheca diameter among artificially reared females. The dopamine levels were significantly higher in females with mandibular notches than those without. In fighting experiments with the intermediate caste females, the winners had significantly higher dopamine levels in the brain than the losers. Brain levels of tyrosine were positively correlated with those of catecholamines but not phenolamines, thereby suggesting a strong metabolic relationship between tyrosine and dopamine. Thus, the caste-specific characteristics of the honey bee are potentially continuous in the same manner as those in primitively eusocial species. Dopamine production in the brain is associated with the continuous caste-specific morphology, as well as being linked to the amount of tyrosine taken from food, and it supports the aggressive behavior of queen-type females. Caste polymorphism in eusocial insects is based on phenotypic plasticity to allow specialization in particular tasks by different colony members. The phenotypes are usually divided discontinuously into different morphological and behavioral types in advanced eusocial species, whereas those in primitively eusocial species are continuous with overlapping morphology between workers and queens, including body size –4. UnderIn advanced eusocial Hymenoptera, including honey bees, the female caste is determined by nutritional states in the early larval stage. In female honey bee larvae, feeding on royal jelly during the first three larval instars leads to a queen phenotype ,5. SugarDopamine is a biogenic amine that functions as a neurotransmitter, neuromodulator, and neurohormone in the central and peripheral nervous systems of insects –13. In tIn contrast to the caste-specific external morphology and some internal morphological features determined during the late larval and prepupal stage in advanced eusocial insects –35, the The artificial rearing of female honey bee larvae is a powerful technique for investigating the mechanisms that might underlie caste differentiation –42. ThisApis mellifera) colonies. The larvae were each transferred into a plastic queen cup with an artificial diet   and phenolamines (tyramine and octopamine) in insects [N-acetyldopamine, tyramine, and octopamine in the brains. In addition, we analyzed the same brain samples using another HPLC-ECD system to quantify the levels of tyrosine and DOPA in the brains.Tyrosine is a common potential precursor of catecholamines [Dihydroxyphenylalanine: DOPA (a precursor of dopamine), dopamine, and  insects ,51. To dThe HPLC-ECD system for analyzing tyrosine and DOPA developed by  comprisemax, IMSmin, spermatheca diameter, and the dopamine levels in the brain were compared among regular workers, control females, 1.5×fed females, and regular queens using the Kruskal–Wallis test with the Steel–Dwass multiple comparison test (significant differences were accepted at P = 0.05). The proportions of individuals with a mandibular notch and different numbers of ovarioles were compared among the groups using Fisher’s exact test with Holm’s correction.Parameters comprising IMSmax, IMSmin, or the spermatheca diameter were examined based on Spearman’s rank correlation coefficients. Associations between brain dopamine levels and differences in the numbers of ovarioles were compared using the Kruskal–Wallis test with the Steel–Dwass multiple comparison test. The dopamine levels in the brains of winners in the fighting experiment were compared with those of losers and non-fighting females using the Steel multiple comparison test . The correlations between the brain levels of tyrosine and those of catecholamines and phenolamines were examined based on Spearman’s rank correlation coefficients.For the artificially reared females, the dopamine levels were compared between females with or without a mandibular notch using the Mann–Whitney U test. Correlations between the dopamine levels with IMSP < 0.001, P < 0.01). The IMSmax values differed significantly among the four groups , with the largest values in queens and the smallest in workers , but it differed only marginally from that in queens (P = 0.06). The number of ovarioles in control females did not differ significantly from that in workers (P = 0.351) but it was lower than those in 1.5×fed females and queens (P < 0.001). The spermatheca diameter differed significantly among workers, control females, 1.5×fed females, and queens , where the diameter was largest in queens but smallest in workers . The spermatheca diameter was significantly larger in control females than workers but smaller than those in 1.5×fed females and queens (P < 0.05).There were clear caste-specific differences in the morphology of the ovaries and spermatheca. Females reared artificially possessed intermediate characteristics between regular workers and queens reared naturally. The number of ovarioles differed significantly among workers, control females, 1.5×fed females, and queens . The dopamine level was significantly higher in 1.5×fed females than control females (P < 0.05), and intermediate between those in workers and queens. The dopamine level was significantly lower in control females than queens (P < 0.05), but not different from that in workers.There were clear caste differences in dopamine production as previously reported ,16. FemaP < 0.001, max values were positively correlated with the dopamine levels in the brain  in the brains than workers [N-acetyldopamine during the pupal stage. Thus, the larval nutritional state may be linked to the intake of tyrosine to influence the brain level of dopamine at emergence and caste-specific behavior including aggression during the early adult stage in honey bee females.The brain dopamine levels in the intermediate caste females at emergence might have been influenced greatly by the amount of the dopamine precursor tyrosine in the brain. Our results showed that the brain levels of tyrosine were positively correlated with the levels of DOPA, dopamine, and  workers . However workers –60, alth workers . The int workers ,62, and N-acetyldopamine, thereby suggesting a strong metabolic relationship between tyrosine and dopamine in the brain. Thus, caste-specific characteristics are potentially continuous depending on the amount of food provided, and this property may be conserved among primitive and advanced eusocial bees. The dopamine level in the brain is also a caste-specific characteristic linked to the nutritional state during the larval stage and it may be associated with aggressive behavior in queen-type females.In this study, we determined whether caste-specific discontinuous characteristics of the external and internal morphology, brain dopamine level, and aggressive behavior can be made continuous by artificially rearing with different amounts of diet. The morphological characteristics of the mandible and reproductive organs, as well as the levels of dopamine in the brain, were discontinuous between workers and queens reared naturally in the nest. However, females given 1.5 times the amount of control diet possessed morphological characteristics intermediate between castes. These females also had intermediate levels of dopamine in the brain. In fighting experiments, the winners had higher dopamine levels in the brain than the losers. The brain levels of tyrosine were positively correlated with those of DOPA, dopamine, and S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Molecular coupling defines fundamental properties of materials, yet spectroscopic access and imaging have remained challenging due to the short length scales of order and disorder and the low energy scales of interactions. We employ vibrational excitons as local structural probes in nanoscale infrared imaging and spectroscopy to map molecular order in a model organic electronic material (octaethyl porphyrin ruthenium(II) carbonyl). We observe coexistence of both amorphous and crystalline phases. Even for highly crystalline porphyrin, the size of individual ordered regions can remain limited to a few molecular lengths. This approach of vibrational exciton nanospectroscopy is broadly applicable to many interacting molecular systems and can provide insight into structure and energy transfer in organic electronics, proteins, or other biological complexes. s-SNOM) with complementary small-angle X-ray scattering to map multiscale structure from molecular coupling to long-range order. In the model organic electronic material octaethyl porphyrin ruthenium(II) carbonyl (RuOEP), we observe the evolution of competing ordered and disordered phases, in nucleation, growth, and ripening of porphyrin nanocrystals. From measurement of vibrational exciton delocalization, we identify coexistence of ordered and disordered phases in RuOEP that extend down to the molecular scale. Even when reaching a high degree of macroscopic crystallinity, identify significant local disorder with correlation lengths of only a few nanometers. This minimally invasive approach of vibrational exciton nanospectroscopy and -imaging is generally applicable to provide the molecular-level insight into photoresponse and energy transport in organic photovoltaics, electronics, or proteins.Much of the electronic transport, photophysical, or biological functions of molecular materials emerge from intermolecular interactions and associated nanoscale structure and morphology. However, competing phases, defects, and disorder give rise to confinement and many-body localization of the associated wavefunction, disturbing the performance of the material. Here, we employ vibrational excitons as a sensitive local probe of intermolecular coupling in hyperspectral infrared scattering scanning near-field optical microscopy (IR  Intermolecular coupling and associated nanoscale structure are intrinsically linked to macroscopic properties from optical and electronic response to biological or catalytic function –5. Espec612However, fundamental understanding and control of the underlying microscopic processes has remained limited due to the low-energy scales of intermolecular interactions and associated wave-function delocalization. Yet, established X-ray or electron spectroscopy and imaging for understanding atomic and molecular order with high spatial resolution rely on high-energy photons or electrons, at fluences often not compatible with delicate organic materials, and lacking sensitivity to the low-energy scales of intermolecular interactions –19.Vibrational resonances as intrinsic molecular labels are uniquely sensitive to the low-energy scales of intermolecular coupling and molecular disorder. In particular, vibrational excitons can form and delocalize on nanometer-length scales  and can 21s-SNOM) and vibrational nanospectroscopy –30s-SNOMtroscopy B as a retroscopy , 32, we This approach of vibrational exciton nanospectroscopy and -imaging is generally applicable to resolve many of the molecular interactions that define the properties of the wide range of molecular electronic, photophysical molecular, or hybrid inorganic–organic materials. It complements conventional imaging and spectroscopy methods to provide a deeper fundamental understanding of low-energy intermolecular interactions, molecular assembly, and ordering at the heart of the structure–function relationship in molecular materials.s-SNOM, as shown in −1, optimized with −1 bandwidth for maximal spectral irradiance and sensitivity. As illustrated in −1 spectral resolution by using lock-in demodulation at the second harmonic of the cantilever motion  (P3HT) B, with mr motion , 33  variation in line width between aggregates. While previous far-field studies have attributed this spectrally broad feature to isolated RuOEP molecules within the P3HT matrix . Under the approximation of uniform transition dipole moment strength of the metal-carbonyl peak in both phases  C, as exps-SNOM determined by the We next imaged an RuOEP aggregate with high crystallinity. Nanoscale voxel array measured with 20-nm grid spacing A and cors-SNOM spectra in the −1. In regions with higher crystallinity We then analyzed for variations in peak position associated with crystallinity. −1 and −1. Regions with a low crystalline fraction −1, whereas regions with high crystalline fraction −1. We identified the maximum peak shift with −1 for highly ordered nanocrystals annealed for 180 min and the minimum peak shift with −1 from crystals with low −1 . This shift of −1. Extending this model to two molecules per unit cell, consistent with known structures of RuOEP polymorphs  coupled to an IR microscope  and a water-cooled SiC globar source. The microscope aperture was set to −1) to 15 −1), with a pulse duration of 150 fs. The laser-output frequency was tuned to that of the carbonyl vibration, with a bandwidth of 100 cm−1. Sixteen milliwatts of DFG light was attenuated by 75% with a mesh filter and was directed into the s-SNOM instrument  as described previously  from a femtosecond OPO  pumped by a low-noise Yb oscillator operating at 75.7 MHz, with a pulse duration of 93 fs and an average power of 6 W . The DFG light was tunable from 4 eviously . Active s-SNOM spectra were collected with 2-cm−1 spectral resolution by using rapid-scan detection and averaging times of 1 to 4 min.Incident laser light was focused by an off-axis parabolic mirror  onto the metallized tip  of an atomic force microscope  operating in intermittent contact (tapping) mode . The neaectively , 33. IR SI Appendix. Data, computer code for fitting, and modeling code are available through the Open Science Repository (https://osf.io/72vcx/) (The procedures of experiments and simulations are described in detail in /72vcx/) .Supplementary File


A 36-year-old man, HIV-infected for the past eight years, non-adherent to antiretroviral therapy (ART), was admitted to the emergency department with fever, low back pain, asthenia, and dyspnea associated with hyaline-secretive cough. Physical examination showed violaceous lesions on the upper trunk and soft palate. Biopsy confirmed the histopathological diagnosis of Kaposi sarcoma (KS). Chest radiography  showed nHigh-resolution chest computed tomography (CT)  revealedThese clinical manifestations highlight the potentially aggressive course of KS in HIV-infected patients. linical evaluation with staging of HIV-related KS may determine the future treatment course. Characteristic CT findings in AIDS-related KS include peribronchovascular and interlobular septal thickening, bilateral and symmetric ill-defined nodules in peribronchovascular distribution, fissural nodules, mediastinal adenopathies, and pleural effusions