Acoustic cavitation was detected using a weakly focused passive sensor. Tumour dimensions were measured with B-mode ultrasound before treatment and with callipers post-mortem. Immune cell subtypes were quantified with immunohistochemistry and flow cytometry. pHIFU treatment of pancreatic tumours resulted in detectable acoustic cavitation and increased infiltration of CD8+ T cells in the tumours of pHIFU and pHIFU + ICI-treated subjects compared with sham-exposed subjects. Survival of subjects treated with pHIFU + ICI was extended relative to both control untreated subjects and those treated with either pHIFU or ICI alone. Subjects treated with pHIFU + ICI had increased levels of CD8+IFNγ+ T cells, increased ratios of CD8+IFNγ+ to CD3+CD4+FoxP3+ and CD11b+Ly6G+ cells, and decreased CD11chigh cells in their tumours compared with controls. These results provide evidence that pHIFU combined with ICI may have potential for use in pancreatic cancer therapy.Pulsed high-intensity focused ultrasound (pHIFU) uses acoustic pressure to physically disrupt tumours. The aim of this study was to investigate whether pHIFU can be used in combination with immune checkpoint inhibitors (ICIs) to enhance survival of tumour-bearing animals. Murine orthotopic pancreatic KPC tumours were exposed both to a grid of pHIFU lesions (peak negative pressure = 17 MPa, frequency = 1.5 MHz, duty cycle = 1%, 1 pulse s The majority of patients are diagnosed at an advanced stage of disease, too late for the surgery that is their only curative treatment option and that is only available to approximately 20% of patients . Even afAdvances in cancer immunotherapy have led to antibodies (immune checkpoint inhibitors (ICIs)) approved by the US Food and Drug Administration for the treatment of several cancers including melanoma and lung cancer –12. ICIsHigh-intensity focused ultrasound (HIFU) is a physical modality that uses non-invasive focused acoustic beams  and thatP−) = 1.65 MPa, frequency (f) = 1 MHz, 0.1 ms long pulses) was combined with microbubbles and anti-PD-1 antibodies [−3) Neuro2A tumours were treated with higher rarefactional pressure exposures  in combination with anti-CTLA-4/PD-1 antibodies and was associated with the induction of systemic immunity including the pro-immune regulation of natural killer cells, CD11c+ cells and circulating cytokines [P− = 30 MPa, f = 1 MHz, 50 pulses) capable of improving tumour growth control in their own right [Some indication of the potential of combining pHIFU and ICI has come from eloquent studies in subcutaneous models of colorectal , neurobltibodies . By contytokines . The relwn right . Thus, iWe predict that if pHIFU can mechanically disrupt tissue , then, i. 2. 2.1KrasG12D/+; Trp53R172H/+; Pdx-1-Cre) supplied by Prof. Tuveson  were used to establish syngeneic orthotopic tumours in C57BL/6J (000664) subjects [2 flasks in a humidified atmosphere containing 5% CO2. Screening for mycoplasma contamination was carried out before transplantation. Orthotopic KPC tumours were established by injecting 105 KPC cells of low passage number (less than 3) in 10 µl of Matrigel into the pancreas (laparotomy) of immune-competent subjects (greater than 15 weeks old), as described in electronic supplementary material, S(a).Murine pancreatic cancer KPC cells ; (B) pHIFU treatment group—exposed to pHIFU using a small animal system   = 1%, exposure time = 25 s) . In order to avoid damage to adjacent organs, only the central portion of the tumour was exposed. To exclude the possibility of wrongly identifying and targeting neighbouring tissues, the superior and inferior tumour edges were first identified under B-mode ultrasound imaging guidance. The tumours were then exposed to pHIFU under ultrasound guidance using a grid of horizontal and vertical lesions 2 mm apart (figure 1c). No lesion was placed within 2 mm of the tumour's superior or inferior edge or within 1 mm of its lateral edges. The number of pHIFU exposures for each tumour thus depended on the tumour dimensions and on the ability to differentiate tumour from neighbouring tissues. Subjects in this group were injected IP with the standard regime of isotype antibodies described above for the control group (figure 1a); (C) ICI treatment group—sham exposed to pHIFU and injected IP with the anti-CTLA-4/anti-PD-1 antibodies (200 µg/antibody/dose/mouse) ; and (D) combined pHIFU + ICI treatment group—treated with the pHIFU and anti-CTLA-4/anti-PD-1 antibodies as described above.Subjects carrying orthotopic pancreatic tumours were randomly assigned to one of the four groups: (A) control group—sham-exposed to pHIFU to reveal any potential physiological effects of anaesthesia, handling and partial submersion in degassed water, and injected intraperitoneally (IP) with rat monoclonal IgG2A and mouse IgG2B isotype antibodies at the same concentration (200 µg/antibody/dose/mouse) and frequency (every 3 days) as the ICIs used in treatments, in order to mimic non-specific activation of the immune system (figure 1a).For tumour growth and immunophenotype analysis experiments (12 day experiments), 24 animals were used, 6 per experimental group. For survival experiments (up to 21 days), another 24 subjects were used , with three separate experiments being carried out for each group. Eleven animals were used for acute experiments where immune phenotype analysis was carried out in tumours and lymphoid organs 48 h after treatment a.. 2.3Tumours were imaged in the cranial/caudal  and medial/lateral  imaging planes using two-dimensional high-frequency (14 MHz) B-mode ultrasound . Images were recorded in planes 1 mm apart in order to obtain tumour dimensions in three orthogonal directions. At the time of culling, tumours were excised and their dimensions measured with Vernier callipers. For survival studies, subjects were culled when their tumour reached 300% of its initial volume (i.e. that measured 1 day before the pHIFU or sham exposure). Tumour volume was calculated assuming an ellipsoidal shape using. 2.4d). Cavitation data acquisition details have been published previously [Acoustic cavitation was monitored during pHIFU bursts using a weakly focused broadband (BB) polyvinylidine fluoride (0.1–20 MHz) passive cavitation detector (PCD) transducer . This was aligned with the pHIFU focal peak at the start of each treatment session using a pulse–echo technique d. Cavitaeviously , and areeviously . In addi. 2.5+ and tumour cells were quantified automatically in whole tumour sections using the instructions and parameters shown in electronic supplementary material, tables S4 and S5. ImageJ was also used to determine the number of positively stained CD8+ cells in high-magnification tumour images and to provide their positional information in whole tumour sections as described in electronic supplementary material, S(d).Excised tumours were formalin-fixed, paraffin-embedded and histologically stained with haematoxylin and eosin (H&E), Masson's trichrome stain and immunohistochemistry (IHC) antibodies. Details of these methods, the antibodies used and acquisition of images can be found in electronic supplementary material, S(c). To quantify this signal, ImageJ analysis software was used ,34 for o. 2.6g. To obtain a tumour cell fraction, tumours were cut into 3 mm pieces and enzymatically disintegrated for 1 h at 37°C using 0.2 Wunch units ml−1 of Liberase; the remaining intact tissue was further mechanically dissociated using a tissue grinder. Cells derived from tumour, spleen and TDLNs were counted with a haemocytometer and 105 cells were transferred to flow cytometry tubes for staining. Cells were incubated with blocking buffer /1% fetal calf serum containing 1 : 100 dilution of anti-CD16/CD32 antibody ) for 1 h to block non-specific binding sites, and then incubated for 5 min with 4′,6-diamidino-2-phenylindole (DAPI) (3 µM) at 4°C to differentiate between live and dead cells. For staining of cell surface receptors, cells were immediately stained with primary antibodies conjugated to a fluorochrome for 1 h (1 : 100) at 4°C in the dark. For the staining of FoxP3, cells were fixed and permeabilized with the FoxP3 fixed and permeabilization buffer (Thermofisher Scientific Ltd) for 40 min. Then cells were washed twice with the permeabilization buffer provided in the kit and stained with (1 : 100 in permeabilization buffer) FoxP3 antibody for 1 h at room temperature. Then cells were washed twice with permeabilization buffer and then with PBS. Signal was detected immediately using a FACSCalibur LSRII . Murine live cells, T cells, cytotoxic T cells, regulatory T cells, myeloid-derived suppressor cells (MDSCs) and dendritic cells were defined as DAPI−, CD3+, CD3+CD8+, CD3+CD4+FoxP3+, CD11b+Ly6G+ and CD11c+, respectively [For analysis of immune biomarkers, the pancreaticoduodenal lymph nodes (TDLNs) draining the spleen and tumour were identified , excised and mechanically disintegrated to obtain single-cell suspensions through a 40 µm mesh filter. The cell fraction was collected after centrifugation for 5 min at 200. 2.7−1) were added to quantify the test signal. After incubation for 2 h at room temperature, the supernatant was removed and plates were washed five times with the wash buffer provided in the kit. Plates were incubated with the capture antibody for 2 h at room temperature and then washed five times with the wash buffer. Plates were incubated with avidin–horseradish peroxidase for 30 min at room temperature, washed and then incubated with tetramethylbenzidine substrate solution for 15 min at room temperature. Stop solution was pipetted into the wells to end the development of the signal. Signal was assessed using a colorimetric plate reader at 405 nm with a reference wavelength at 495 nm.The Ready Set Go ELISA kits (E-bioscience/Affymetrix) were used to detect interferon-γ (IFNγ) and tumour necrosis factor-α (TNFα) in the blood plasma of subjects. Briefly, wells of a 96-well microwell plate were incubated overnight with IFNγ or TNFα biotin-conjugated antibody at 4°C. The next day the wells were washed five times with the wash buffer provided in the kit. Fifty microlitres of blood plasma/PBS mixture obtained from each subject was pipetted into each well. Alongside these wells, purified IFNγ and TNFα in increasing concentrations , and a collagen-containing microenvironment surrounding the tumour and the intercellular space . Tumours were visible under ultrasound imaging before and after pHIFU treatment (figure 1f). The insonated volumes in the pHIFU and the pHIFU + ICI treatment groups covered 5.3 ± 4% and 5.2 ± 3.8% of the whole tumour, respectively (figure 1g) and resulted in lesion-like (disrupted) areas which covered 9.5 ± 4.8% and 11.9 ± 8% of the whole tumour volume, respectively, 12 days after pHIFU treatment. These regions had exhibited lower (or zero) cell densities and were also seen in the subjects of the control and ICI groups  possibly because of physiological necrosis of the tumour's core (figure 1h). The disruption of insonated tumours was evident immediately after pHIFU treatment. Haematomas were present, and disruption was sustained, as shown in pictures of tumours stained with H&E immediately, 6 and 12 days after treatment (figure 1i). We then investigated whether tissue disruption seen in pHIFU-treated subjects was associated with acoustic cavitation.Our goal was first to characterize the baseline effects of pHIFU treatments on KPC tumours. Subject randomization and resulting initial group tumour volumes and dimensions for the survival and 12 day experiments are shown in figure 1. 3.2a,b. This shows continuously elevated HH emissions and frequently elevated BB signals (relative to the reference frequencies) that are additionally significantly above off-time levels. In addition, there was drive voltage kurtosis (e.g. around 2 and 4 ms) . Acoustic cavitation was seen in all subjects in the pHIFU and pHIFU + ICI groups. When normalized for tumour volume, subjects in the pHIFU and pHIFU + ICI groups showed HH signals of 510 ± 350 µV s mm−3 and 550 ± 400 µV s mm−3, respectively. Subjects in the pHIFU and pHIFU + ICI group showed BB signals of 120 ± 200 µV s mm−3 and 150 ± 200 µV s mm−3, respectively (figure 2c). These differences in the acoustic cavitation signal between the pHIFU and pHIFU + ICI groups were not statistically significant. Also, no statistically significant differences in HH and BB emissions/insonation volume were seen between pHIFU and pHIFU + ICI tumour treatments. Subjects treated with pHIFU, randomized into the pHIFU and pHIFU + ICI groups, showed HH signal/insonation volumes of 10 000 ± 6000 µV s mm−3 and 12 000 ± 7500 µV s mm−3, respectively, and BB signal/insonation volumes of 2600 ± 5000 µV s mm−3 and 2300 ± 3500 µV s mm−3, respectively (figure 2d). These results suggest that all pHIFU treatments induced acoustic cavitation, leading to mechanical disruption of tumours in both pHIFU and pHIFU + ICI groups. This led us to investigate whether survival could be increased using these treatments.A representative example of the PCD signal obtained during a 10 ms HIFU pulse is shown in figure 2. 3.3. 3.4+ lymphocytes in tumours, their abundance was assessed 2 and 12 days after treatment. Representative images of tumours of all subjects showed positive staining of CD8+ TILs 12 days after treatment. On average, images of control, pHIFU-treated, ICI-treated and pHIFU + ICI-treated tumours had 24 ± 22, 54 ± 32, 28 ± 15 and 55 ± 32 CD8+ cells per image, respectively (a). When whole tumour sections were assessed, a statistically significant increase in their abundance was detected in subjects in the pHIFU and pHIFU + ICI groups compared with those in control and ICI groups. In these tumour sections, the number of CD8+ T cells in the control, pHIFU, ICI and pHIFU + ICI groups (expressed as a percentage of the number of tumour cells) were 2.3 ± 1.6%, 4.7 ± 3.0%, 2.3 ± 1.9% and 5.8 ± 4.0%, respectively, 12 days after treatment (b). Furthermore, increased CD8+ TILs were seen within 0.5 mm of the tumour periphery compared with non-peripheral areas (0.5–5 mm from the tumour's edge) in ICI, pHIFU and pHIFU + ICI-treated subjects . A statistically significant increase in the levels of IFNγ, but not TNFα , was detected in the blood of subjects treated with pHIFU (250 ± 80 pg ml−1), ICI (450 ± 200 pg ml−1) and pHIFU + ICI (550 ± 180 pg ml−1) relative to that of controls (180 ± 20 pg ml−1) 12 days after treatment (c). Flow cytometric analysis of whole tumours 48 h after treatment with pHIFU + ICI showed increased levels of CD8+ and CD8+IFNγ+ T cells in these tumours relative to control subjects. Tumours of subjects treated with pHIFU + ICI showed that 12 ± 5% of their live (DAPI−) cells were CD3+CD8+, whereas this was 8 ± 3% in controls. Also, 0.6 ± 0.3% of all live cells in the tumours of pHIFU + ICI-treated subjects were CD8+IFNγ+ T cells, whereas this was only 0.2 ± 0.1% in controls (d). The CD8+IFNγ+/CD4+ T-cell ratio was increased in pHIFU + ICI-treated subjects (0.08 ± 0.04) compared with that in control subjects (0.01 ± 0.005). Also, 2 ± 1% of live cells in pHIFU + ICI-treated tumours were CD11chigh, whereas this frequency was increased in control tumours (4 ± 1.5%) (e). In addition, non-statistically significant differences were seen in the frequency of CD4+FoxP3+ T cells and CD11b+Ly6G+ cells in the tumours of pHIFU + ICI-treated subjects compared with controls 48 h after treatment. The tumours of control and pHIFU + ICI-treated subjects showed that 2 ± 1% and 3 ± 1% of their live cells were CD3+CD4+FoxP3+, respectively, and 7 ± 4% and 10 ± 4% were CD11b+Ly6G+, respectively (f). An increase was detected in the ratios of CD8+IFNγ+/CD4+FoxP3+ T cells and CD8+IFNγ+/CD11b+Ly6G+ cells in pHIFU + ICI-treated tumours relative to controls. These ratios in control and pHIFU + ICI-treated subjects were 0.1 ± 0.05 and 0.25 ± 0.15 (CD8+IFNγ+/CD4+FoxP3+ T cells), respectively, and 0.03 ± 0.02 and 0.06 ± 0.02 (CD8+IFNγ+/CD11b+Ly6G+ cells), respectively (f). Also, 12 days after treatment, FoxP3+ cells were detected, irrespective of the treatment group, only in the periphery of the tumours (g). These results not only show the increased infiltration of CD8+ TILs into mechanically disrupted tumours, but also that the relative frequency of IFNγ+CD8+ T cells over that of other immune regulators including regulatory T cells and MDSCs increases within these tumours.In order to investigate whether the increased cavitation signals, and the improved survival and tumour growth control of subjects in the pHIFU + ICI group were associated with increased infiltration of CD8ectively a. When wreatment b. Furthereatment c. Flow ccontrols d. The CD ± 1.5%) e. In addectively f. An incectively f. Also,  tumours g. These . 3.5+ T cells, but not that of CD8+IFNγ+ T cells, in the TDLNs of pHIFU + ICI-treated subjects relative to controls was observed 48 h after treatment. TDLNs of control and pHIFU + ICI-treated subjects showed that 15 ± 3% and 9 ± 3% of live cells were CD3+CD8+, respectively, and 0.3 ± 0.2% and 0.2 ± 0.1% were CD3+CD8+IFNγ+, respectively (a). The decrease in the abundance of CD8+ T cells in the TDLNs of pHIFU + ICI-treated subjects was associated with a decrease in CD3+ T cells in the TDLNs of pHIFU + ICI subjects and decreased frequency of the CD8+ T cells among the CD3+ cell population. The frequencies of these populations in control and pHIFU + ICI-treated subjects were 46 ± 7% and 33 ± 7% (CD3+ T/live cells), respectively, and 31 ± 6% and 24 ± 5% (CD8+/CD3+ T cells) 48 h after treatment (a). These results suggest that the acute effect of pHIFU + ICI treatments in the tumours is associated with the decreased draining of CD8+ T cells to the TDLNs of subjects.The decreased frequency of CD8ectively a. The dereatment a. These +FoxP3+ T cells in the spleen but not in the TDLNs, of pHIFU + ICI-treated subjects compared with controls 48 h after treatment. Control and pHIFU + ICI-treated subjects had 6 ± 3% and 5 ± 1%, respectively (TDLNs), and 3 ± 1% and 5 ± 1%, respectively (spleens), of live cells that were CD3+CD4+FoxP3+ (b). No statistically significant differences were seen in the frequency of CD11b+Ly6G+ cells, nor in the ratios of CD8+/CD4+FoxP3+ T cells, CD8+/CD11b+Ly6G+, CD8+IFNγ+/CD4+FoxP3+ and CD8+IFNγ+/ CD11b+Ly6G+ in the spleens and TDLNs of control and pHIFU + ICI-treated subjects . To investigate whether the regulation of CD4+FoxP3+ T cells in the spleen and TDLNs is sustained longitudinally, their frequency was also investigated 12 days after treatment. Results showed a statistically significant increase in their frequency in the TDLNs of ICI-treated subjects compared with control and pHIFU + ICI-treated subjects (c), but not in their spleens . The percentage of these cells in the TDLNs of control, pHIFU, ICI and pHIFU + ICI-treated subjects were 3 ± 2%, 5 ± 2%, 6 ± 1% and 4 ± 2% of live cells, respectively, whereas the CD8+/ CD4+FoxP3+ T-cell ratios were 8 ± 5%, 6 ± 4%, 3 ± 1% and 6 ± 3%, respectively (c). In addition, ICI-treated subjects showed an increased frequency of CD11b+Ly6G+ cells (12 ± 6%) in their spleens relative to control and pHIFU + ICI-treated subjects  12 days after treatment (c).A statistically significant increase was seen in the abundance of CD44+FoxP3+ b. No stasubjects c, but noectively c. In addreatment c.. 4+ and CD8+IFNγ+ TILs, increasing the ratio of CD8+IFNγ+ T cells to CD4+ T cells, regulatory T cells and MDSCs, while controlling the frequency of the last two immune cell types in the tumours to basal levels.The aim of this study was to investigate whether combination treatments involving focused ultrasound and antibody immunotherapy might provide therapeutic potential for use in pancreatic cancer therapy. A murine orthotopic model which recapitulates some hallmarks of the human disease  did not . These data suggested that pHIFU exposures designed to induce mechanical damage in tissue in the absence of significant thermal change may be better equipped to create a pro-inflammatory environment than thermally ablative continuous HIFU treatments which are likely to heat fix target tissue, thus limiting intratumoral antigen release [P− = 17 MPa, d.c. = 1%, 10 ms exposures) that were considered ‘safe’ were chosen to avoid the induction of non-specific inflammation since treatment with higher pulse negative pressures (P− ≥ 22 MPa), and attempts to ablate the whole tumour resulted in some skin and spleen damage. The dimensions of the focal volume of the ultrasound beam were 5 mm axially and 1 mm transversely. Therefore, to avoid damaging adjacent organs, tumours were treated when they were longer than 5 mm on axis. This resulted in a non-statistically significant variability in tumour dimensions and volume , nor was there a difference between these two groups in the insonated tumour volumes (g). These results show that acoustic cavitation, suggestive of mechanical disruption and/or destruction of the cells and tissue, is induced in all pancreatic tumours treated with pHIFU and pHIFU + ICI, and that the anti-cancer benefit observed in pHIFU + ICI-treated subjects over the pHIFU-only treated subjects is not a consequence of technical variability during pHIFU exposures.All pHIFU exposures resulted in detectable acoustic cavitation. HH emissions, indicative of the activity of gas microbubbles inside the tumour which could disrupt and permeabilize the cells and tissues, were observed in all tumours. In addition, BB activity, suggestive of inertial cavitation, which could result in increased levels of destruction of the exposed volume, was detected in 10 of 12 tumours in each of the pHIFU and pHIFU + ICI groups. This is the only study on the effects of pHIFU and immunotherapy so far to document acoustic cavitation and thus verify the mechanical effect inside every treated tumour. This is of particular relevance in deep-seated orthotopic tumours where some attenuation of the focused ultrasound signal is to be expected. No statistically significant differences in cavitation emissions between pHIFU and pHIFU + ICI tumour treatments were seen c,d, nor  volumes g. These e). Anti-CTLA-4 was combined with anti-PD-1 as evidence suggests that the anti-CTLA-4 and anti-PD-1 blockades are conferred by different cellular mechanisms [Subjects were treated with a PD-1 ICI to abrogate the immune inhibitory effects of the PD-1–PD-L1 axis since KPC cells were found to express PD-L1 e. Anti-Cchanisms ,42. Our chanisms ,44. Usinchanisms , and if + TILs. To test this, TILs were investigated in tumours that were subjected to acoustic cavitation (pHIFU and pHIFU + ICI groups) and in tumours that were sham exposed (control and ICI groups) 12 days after treatment. IHC analysis was used instead of flow cytometry in order to avoid the extensive mechanical and enzymatic processing of relatively large and stiff pancreatic tumours (e.g. control group), and to provide positional information for the cells. No significant advantage of one technique over the other has been shown for biomarker analysis in previously published studies [+ T cells (b) relative to the control and ICI groups, suggesting that the cavitation induced by pHIFU allowed increased access to the tumours for these cells or created an environment which had a greater recruitment signal for antigen-activated T cells. However, expression of PD-L1 by the KPC cells (e) would be expected to limit their activation. To overcome this resistance, ICIs have been used, resulting in a therapeutic benefit in the pHIFU + ICI treatment group that was not seen in the pHIFU group (where no ICI were used). These results explain, at least partly, the beneficial anti-cancer effect seen in pHIFU + ICI subjects relative to all other treatment groups, and suggest that the mechanical disruption induced by pHIFU resulted in sustained elevated levels of CD8+ TILs in the tumours. When coupled with anti-PD-1 to abrogate the PD-1/PD-L1 inhibitory axis, and anti-CTLA-4 to maximize immunotherapeutic potency , enh+ TILs and CD8+IFNγ+ TILs (d), raised the ratio of CD8+IFNγ+ TILs to regulatory T cells, MDSCs and CD4+ T cells  and retained the levels of regulatory T cells and MDSCs at the levels seen in control subjects (f). Also in murine, but not human, subjects, the reduction of cell surface CD11c staining (as seen here in pHIFU + ICI-treated tumours) (e) has been associated with the activation of dendritic cells [c).Tumour growth depends on the balance between pro- and anti-immune regulators . Our resNγ+ TILs d, raised T cells e,f and rsubjects f. Also itumours) e has beeic cells . These ric cells ,19. Fina+ T cells seen in TDLNs (a) raises the question of whether this is due to the disruption of the tumour's lymph vessels by the ultrasound treatment. The disruption of the vasculature leading to decreased blood flow has been associated with the anti-cancer effects of pHIFU + anti-PD-1 [c), and the possibility that these could be indicative of an adverse systemic inflammatory response cannot be excluded. Cell depletion experiments (e.g. CD8 and/or IFNγ) would prove conclusively whether such a regulation exists. pHIFU-ICI-treated tumours show at least some irregular areas of collagen depletion and reorganization when compared with the dense-collagen microenvironment of control tumours . In order to design the most effective treatments, there is thus a need to increase our understanding of the effects of these treatments on the basic biological resistance mechanisms exploited by pancreatic ductal adenocarcinoma (PDAC). For example, differentiating between apoptotic (including immunogenic cell death) and acute necrotic events could show for how long the anti-cancer effects of the treatments could be sustained, and provide further explanation for the activation of the immune system. The use of repeated pHIFU treatments with the objective of augmenting the anti-tumour response using clinically feasible treatment parameters in genetically engineered PDAC subjects, and the application of modern high-throughput techniques  would aid those studies. In addition, longer endpoints would make possible the longitudinal characterization of the tumoral microenvironment, including the regulation of cancer-associated fibroblasts and macrophages [The data presented in this study give rise to questions the answers to which would provide further insights into the effects of the pHIFU and ICI combinations in pancreatic cancer. For example, a reduction in the frequency of CD8in TDLNs a raises nti-PD-1 . Such merophages  and a st. 5A physical modality, focused ultrasound, has been used in combination with immunotherapy, in the form of ICI antibodies, to contain disease progression in KPC pancreatic tumour-bearing animals. A step-by-step justification for our pHIFU targeting decisions in order to mechanically disrupt these orthotopic tumours, without endangering the animal, has been given. The pHIFU-induced mechanical disruption of the tumours has been traced by detecting acoustic cavitation signals for each animal. Animals that were exposed to pHIFU + ICI survived longer than sham-exposed subjects or subjects treated with focused ultrasound or ICI alone. This improvement in disease progression in pHIFU + ICI subjects compared with control subjects has been explained by the increase of TILs in mechanically disrupted tumours, and the creation of a localized tumoral pro-inflammatory microenvironment when coupled with ICI. These results indicate that focused ultrasound may be able to potentiate the anti-cancer effects of traditional oncological interventions such as immunotherapy, and is a promising candidate for clinical use.


These states are generally mutually exclusive and modulation of one state influences the pattern of the other. Here, we identify the zinc-finger protein OEF-1 as a novel modifier of this epigenetic balance in the C. elegans germline. Loss of oef-1 genetically enhances mes mutant phenotypes. Moreover, OEF-1 binding correlates with the active modification H3K36me3 and sustains H3K36me3 levels in the absence of MES-4 activity. OEF-1 also promotes efficient mRNA splicing activity, a process that is influenced by H3K36me3 levels. Finally, OEF-1 limits deposition of the silencing modification H3K27me3 on the X chromosome and at repressed autosomal loci. We propose that OEF-1 might act as an intermediary to mediate the downstream effects of H3K36me3 that promote transcript integrity, and indirectly affect gene silencing as a consequence.To ensure stable transmission of genetic information to the next generation, germ cells frequently silence sex chromosomes, as well as autosomal loci that promote inappropriate differentiation programs. In Caenorhabditis  Caenorhabditis elegans, these modifications are established via the chromatin-modifying pathway consisting of the histone methyltransferase (HMT) MES-4 and the Polycomb Repressive Complex 2 (PRC2) orthologs MES-2/3/6  as a novel, germline-specific nuclear factor that is required for a normal rate of progression through germ cell development . Germ cemes-4 mutants. Moreover, loss of OEF-1 activity results in reduced splicing efficiency of germline-expressed mRNAs. These observations together suggest that OEF-1 might assess or interpret H3K36me3 levels to mediate downstream functions such as splicing that are associated with productive gene expression. We hypothesize that this activity would indirectly affect gene repression by disrupting the balance between activating and silencing modifications.We first determine that OEF-1 has minimal effect on transcript abundance in germ cells, with mild reduction of X-linked transcript abundance. Consistent with this observation, loss of OEF-1 increases the silencing mark H3K27me3 throughout the genome, particularly on the X chromosome. However, OEF-1 binding primarily correlates with the presence of the activating histone modification H3K36me3, and OEF-1 is necessary to prevent H3K36me3 levels from decreasing in glp-4 strains which were maintained at 15°C and shifted to 25°C before collection. For balanced strains, larval stage 4 (L4) worms expressing pharynx GFP were picked to ensure the selection of first-generation homozygotes in downstream assays.Strains used in this study are listed in Supplementary Table S7. Strains were maintained by standard methods on NGM plates seeded with OP50 bacteria as described . BristolL4 worms were singly placed on seeded plates and moved to fresh plates each day until embryo production ceased. Unhatched embryos were scored as dead 24 h after the mother was removed. Live larvae were counted two days later. To score the presence or absence of germlines, L4s were aged 8–9 h before fixation in 20 μl of Carnoy's fix  on Super Frost slides. Twelve microliters of 1 ng/mL DAPI diluted in M9 was added to coverslips, which were touched to slides and sealed with nail polish before scoring.Adults 16–20 h post-L4 were dissected to release gonads in 1.1x egg salts  with 0.1% Tween 20 and 0.3 mM levamisole. Gonads were fixed with 1% formaldehyde, then freeze-cracked on a metal block embedded in dry ice and immediately placed in −20°C methanol. Blocking was performed using 0.5% BSA in PBST for 30 min at room temperature. For histone modification staining, the area around the gonads was dried with a Kim wipe and a circle was drawn around the tissue using a mini PAP pen before the addition of the primary antibody. Primary and secondary antibody incubations were performed overnight at 4°C in a 50 μl volume. The following antibody dilutions were used: 1:50 anti-OEF-1 ; 1:2000 oef-1(vr25) germ cells. The mean intensity of H3K27me3 in the outlined areas was recorded. The measurements were repeated for 6–8 nuclei per gonad, 7 gonads per genotype .For H3K27me3 mean intensity measurements, ImageJ (NIH) was used to make Z projection images. The X chromosome was identified in germ cells by lack of H3K4me2 staining and enrichment for H3K27me3 staining. In each germ cell in which a clear X chromosome could be identified, a representative autosome was identified by high levels of H3K4me2 and low levels of H3K27me3. The free draw tool was used to outline a representative X and a representative autosome in wild-type and For the H3K36me3 proliferative zone reduction phenotype, extruded and well-stained gonads were scored as having no reduction, slight reduction, or strong reduction in H3K36me3 staining in the proliferative zone as compared to pachytene in the same gonad. The genotypes of the slides were blinded before scoring. The slides represented two or three independent staining experiments all scored in the same sitting.oef-1(vr25) worms were grown to gravid on NGM plates, bleached, and hatched overnight in M9 for at least 24 h. Fifty thousand L1s were plated on enriched plates and grown until most worms had ≥4 embryos. Around 1 million worms were used per nuclei collection. The worms were washed off with M9 in sets of 6 plates into 50 mL falcon tubes, washed twice with M9, then were fixed for 28 min in 2% formaldehyde diluted in M9. After a wash with 1 M Tris (pH 7.5), the worms were washed twice more with M9. The worms were then washed with chilled Nuclei Purification Buffer (NPB) .Germ nuclei isolation was performed as in The fixed and washed worms were resuspended in NPB and transferred to prechilled 7 mL glass Dounce homogenizers (Wheaton). One Dounce homogenizer was used per set of 6 plates. Fifteen loose strokes were followed by 30 tight strokes with a quarter turn between each Dounce. The worms were transferred into chilled 50 mL falcon tubes, and NPB was added to 30 mL. The falcon tubes were vortexed on medium-high speed for 30 s, followed by 15 min on ice. The vortex and ice incubation were repeated. The worms were then passed through three 40 μm filters (Fisherbrand) followed by eight 20 μm filters (pluriSelect). The nuclei were spun at 3100 rpm for 6 min at 4°C. The supernatant was removed, and the nuclei were resuspended in 1 mL NPB and transferred to a nonstick 1.5 mL tube (Ambion). A 5 μl sample of nuclei was removed, incubated with DAPI, and counted using a hemacytometer (Hausser Scientific). Finally, the nuclei were spun at 4°C at 4000 rpm for 5 min, the supernatant was removed, and the nuclei were flash frozen in liquid nitrogen. The nuclei were stored at −80°C until sonication. 2–3 nuclei collections were pooled per ChIP experiment .Isolated germ cell nuclei were thawed on ice, and 120 μl of Nuclear Lysis Buffer  was added to each sample. The nuclei were vortexed vigorously for 1 min followed by 1 min on ice. The vortex step was repeated. The nuclei were sonicated at 2°C in a water bath sonicator (Misonix S-4000) at 20% amplitude, with 10 s on/10 s off pulses for a total process time of 20 min.1.2 mL of FA buffer  was added to each sonicated sample. A 1:20 volume of 20% sarkosyl was added, and the samples were spun at 13,000 × g for 5 min at 4°C. The supernatant was transferred to a new tube, and then 5% of the lysate was removed for the input sample. The input samples were stored at −20°C overnight. Five micrograms of anti-H3K27me3 (Active Motif MABI0323) or anti-H3K36me3 (Active Motif MABI0333) was incubated with each IP sample overnight at 4°C with rotation.The next day, the input samples were thawed on ice and were incubated with 2 μl of 10 mg/mL RNase A for 2 h at room temperature. Approximately 40 μl of Protein G Sepharose beads  were washed 4 times with 1 mL FA, spinning for 2 min at 2500 × g between each wash. The entire IP sample was added directly to the beads, and the samples were rotated for 2 h at 4°C.After the RNase A treatment of the input samples, 230 μl of elution buffer  was added to each input sample to bring the total volume up to 300 μl. 2.05 μl of 19.5 mg/mL proteinase K was added, and the input samples were incubated at 55°C for 3 h.After the 2-h bead incubation, the beads were washed at room temperature as follows (1 mL for each wash): 2 times with FA for 5 min each, 1 time with FA with 500 mM NaCl for 10 min, 1 time with TEL  for 10 min, 2 times with TE for 5 min each. One hundred and fifty microliter of elution buffer was added to each sample and was incubated at 65°C for 15 min with occasional vortexing. The elution step was repeated and the eluates were pooled. 1.03 μl of 19.5 mg/mL proteinase K was added to the eluates and the incubation was performed at 55°C for 1 h. After the proteinase K digestion, input and IP samples were incubated overnight at 65°C to reverse the crosslinks. The next day, input and ChIP DNA were purified using the QIAquick PCR Purification Kit. Samples were eluted in 40 μl TE and submitted for library preparation and sequencing at the Yale Center for Genome Analysis.glp-4 and glp-4; oef-1 animals were grown at the permissive temperature of 15°C and embryos were isolated by bleaching and hatching overnight in M9 at 15°C. Forty thousand L1s were plated on peptone-enriched plates and were grown at 25°C until the young adult stage. Adult worms were washed off plates with M9 and were washed 3 times, spinning at 3100 × g between each wash. Worms were fixed in 50 mL 2% formaldehyde in M9 for 28 min at room temperature. After spinning, worms were washed with 50 mL 1 M Tris, pH 7.5 followed by two more washes with M9. A final wash with FA buffer was performed before pellets were flash-frozen and stored at −80°C. After thawing on ice, pellets were resuspended in 750 μl FA buffer and transferred to a 2 mL Kontes dounce (Kimble Chase). Samples were dounced 15 times for two cycles with the A pestle and 15 times with the B pestle for four cycles with a 1 min hold between each cycle. Pellets were sonicated using a SFX250 sonifier (Branson) in 1.5 mL FA buffer in an ice bath at 22% amplitude with 10 s on/1 min off pulses for a total process time of 5 min 40 s. Sonicated samples were transferred to nonstick tubes and spun at 13,000 × g for 15 min at 4°C. 4.4 mg of lysate was used per IP in a volume of 400 μl. 1:20 volume of 20% Sarkosyl solution was added to each 400 μl sample and the samples were spun at 13,000 × g for 5 min at 4°C. The supernatant was transferred to new nonstick tubes. The rest of the ChIP protocol was performed as for isolated germ nuclei.Somatic samples were prepared as described (https://github.com/guifengwei/glib/blob/master/bam2wig.py), which also scales IP to input using read number. Heatmaps were generated by ngs.plot.r (v2.63) (Seventy-five-bp single-end reads were sequenced on an Illumina HiSeq2500 in rapid-run mode. The raw ChIP and corresponding input fastq sequencing reads were mapped to the genome (version ce10) by Bowtie2 (v2.3.2)  with defoef-1(vr25) worms were grown to gravid on large NGM plates, bleached, and hatched overnight in M9 in the absence of food. The next day, 200 L1s were plated on small NGM plates and were grown to young adulthood. One hundred young adults of each genotype were dissected to release gonads in M9 with 0.1% levamisole and 0.1% Tween 20, cut at the spermatheca, and transferred in as little liquid as possible to 100 μl of TRIzol (Invitrogen) on ice. The samples in TRIzol were flash frozen in liquid nitrogen and were stored at −80°C until subsequent purification steps. Growths and dissections were repeated for a total of 400 gonads per replicate for each genotype.N2 and Samples were thawed in a 37°C water bath, vortexed, and flash frozen again in liquid nitrogen. The freeze-thaw step was repeated twice more. Hundred microliters of chloroform was added, the samples were shaken for 15 s, and then left to sit for 8 min at room temperature. Samples were spun at 12,000 × g for 20 min at 4°C. The top aqueous layer was removed to a fresh tube, then an equal volume of isopropanol was added and incubated at −20°C overnight. The next day, the samples were spun at 12,000 × g for 30 min at 4°C. The supernatant was discarded, and the pellets were washed with 500 µl of 75% ethanol and spun at full speed at 4°C for 5 min. The wash and spin steps were repeated. As much ethanol was removed as possible, and the pellets were dried for 5 min. Finally, the pellets were resuspended with 20 µl of RNase-free water preheated to 55°C. DNase treatment was performed using the Ambion DNA-free kit according to the manufacturer’s instructions. DNase-treated samples were then cleaned up using the RNeasy Mini Kit and eluted in 50 µl of RNase-free water.oef-1(vr25) assemblies was performed using CuffDiff v2.2.1  allele, which contains a 56-bp frameshift deletion in exon 2 generated by CRISPR/Cas9 gene editing (oef-1(vr25) mutants and found relatively few significant changes in gene expression (oef-1(vr25) compared to wild type that was not detected for autosomes using either the CuffDiff or DESeq2 datasets  mutant germ cells compared to wild type (oef-1(vr25) X chromosomes compared to wild type (oef-1(vr25) and wild-type autosomes  adults, and then performed H3K27me3 chromatin immunoprecipitation followed by sequencing (ChIP-seq). As a soma-only control, we also performed H3K27me3 ChIP-seq on glp-4(bn2) and glp-4; oef-1 whole animals, which lack germ cells at the restrictive temperature of 25°C (In order to investigate the changes in H3K27me3 accumulation in  of 25°C . Becauseoef-1(vr25) isolated germ nuclei and found elevated average levels of H3K27me3 on X-linked genes in oef-1(vr25)  mutant germ cells (oef-1(vr25) germ nuclei , 82% are expressed either during spermatogenesis in the L4 stage or in the soma  mutants. As before, we first confirmed that the wild-type H3K36me3 profile reflected the expected pattern for the germline and soma. Indeed, H3K36me3 levels were depleted from the X chromosome relative to autosomes in germ nuclei but not in the soma   . The simdetected . We theroef-1 mutants suggested that OEF-1 might have a genetic interaction with the MES pathway, which establishes these two marks in germ cells. Specifically, because OEF-1 and MES-4 have opposite effects on X-linked H3K27me3, we expected that loss of OEF-1 might suppress the second-generation sterility of mes mutants (oef-1 as well as either mes-4 (the HMT responsible for H3K36me3)  . Abnormal chromatin organization was frequently noted in the double mutant embryos, which might contribute to the lethality . High levels of embryonic lethality also occurred when oef-1 was crossed to a second mes-4 allele (Supplementary Table S3). Consistent with the mes-4 single mutant phenotype, oef-1; mes-4 F2 survivors became sterile adults (Supplementary Table S4). In addition, mes-2; oef-1 first-generation mutants showed a small but significant increase in embryonic lethality  (Supplementary Table S5). mes-2; oef-1 double mutants also exhibited an increase in the incidence of males , suggesting higher rates of X chromosome nondisjunction. These results indicate that loss of OEF-1 has a greater effect in mes-4 mutants relative to mes-2 mutants, consistent with co-localization of OEF-1 and MES-4 at germline-expressed genes . Thus, oef-1 specifically enhances mes-4 but not met-1 defects in H3K36me3 levels.We speculated that the strong genetic interaction between oef-1 with mes-4 in maintaining H3K36me3 levels suggested that OEF-1 might function with other known H3K36me3-associated proteins. We therefore tested whether oef-1 might also have a genetic interaction with mrg-1, the ortholog of another H3K36me3-associated protein, MRG15 . Thus, OEF-1 enhances the phenotypes of two factors associated with H3K36me3, reinforcing the possibility that OEF-1 acts at sites of H3K36me3 in germ cells and might influence H3K36me3-related processes.The relationship of oef-1 mutant gonads, we analyzed the frequency of intron retention, which indicates a disruption of normal splicing events. To rigorously define the baseline variation of intron retention that might normally occur between any two independent samples, we established a control comparison between our wild-type sample and an independently collected wild-type dataset, also from dissected gonads  that might underlie the precocious proliferation and increased apoptosis phenotypes seen upon loss of oef-1. Indeed, these candidate genes display OEF-1 binding . By contrast, genes with more intron retention in the wild type control are biased toward genes expressed in the soma and encode proteins associated with zygotic functions. Finally, a negative correlation exists between transcript abundance and intron retention in oef-1 mutants: genes with decreased transcript abundance are biased toward increased intron retention, whereas genes with increased transcript abundance are biased toward less intron retention . Different H3K36me3 reader protein complexes can affect cryptic transcription initiation, splicing, RNA processing, chromatin remodeling, and DNA repair . Decreased H3K36me3 is not detected in https://doi.org/10.25387/g3.16586714.The gene expression omnibus (GEO) accession number for the ChIP-seq and RNA-seq datasets reported in this paper is GSE147401. Previously published OEF-1::GFP ChIP-seq is available under accession number GSE107190 . Supplem


Phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate phototransduction, and its maturation and function are known to critically depend on a specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Defects in PDE6 and AIPL1 underlie several severe retinal diseases, including retinitis pigmentosa and Leber congenital amaurosis. Here, we characterize the complex of AIPL1 with HSP90 and demonstrate its essential role in promoting the functional conformation of nascent PDE6. Our analysis suggests that AIPL1 preferentially binds to HSP90 in the closed state with a stoichiometry of 1:2, with the tetratricopeptide repeat domain and the tetratricopeptide repeat helix 7 extension of AIPL1 being the main contributors to the AIPL1/HSP90 interface. We demonstrate that mutations of these determinants markedly diminished both the affinity of AIPL1 for HSP90 and the ability of AIPL1 to cochaperone the maturation of PDE6 in a heterologous expression system. In addition, the FK506-binding protein (FKBP) domain of AIPL1 encloses a unique prenyl-binding site that anchors AIPL1 to posttranslational lipid modifications of PDE6. A mouse model with rod PDE6 lacking farnesylation of its PDE6A subunit revealed normal expression, trafficking, and signaling of the enzyme. Furthermore, AIPL1 was unexpectedly capable of inducing the maturation of unprenylated cone PDE6C, whereas mutant AIPL1 deficient in prenyl binding competently cochaperoned prenylated PDE6C. Thus, we conclude neither sequestration of the prenyl modifications is required for PDE6 maturation to proceed, nor is the FKBP-lipid interaction involved in the conformational switch of the enzyme into the functional state. Cyclic nucleotide phosphodiesterases of the sixth family PDE6) are the effector enzymes in both rod and cone photoreceptor cells, whereby light-induced activation of PDE6 generates a hyperpolarizing electrical response propagated to downstream retinal neurons , 2. Rod  are the AIPL1 shares a high sequence identity (∼50%) with AhR-interacting protein (AIP), which serves as cochaperone of HSP90 during the maturation of AhR , 22. AIPA). Addition of AIPL1 to the apo- or the AMPPNP-bound HSP90 led to a shift toward a slower migrating protein complex, suggesting that the cochaperone can bind to both HSP90 states. To assess these interactions quantitatively, we conducted Biolayer Interferometry (BLI) assays using Avi-tagged HSP90 attached to the streptavidin (SA) sensor. These experiments revealed fast association/dissociation kinetics of AIPL1 binding to apo HSP90, yielding a KD (koff/kon) of ∼17 μM . A KD of 32 μM was indicated by the steady-state analysis, reflecting a relatively weak binding of AIPL1 to the open HSP90 state . The binding assay between AIPL1 and the closed HSP90AMPPNP state indicated a significantly higher-affinity interaction and a kinetic and steady-state KD values of ∼2.1 μM and ∼4.5 μM, respectively (C and D).HSP90 chaperones exist mainly as homodimers in conformations ranging from a closed ATP-bound state to open/V-shaped and open-extended states for the apo protein , 36. We Fig. 11–316) (A). Using the BLI assay, the affinity of AIPL11–316 for the closed HSP90 state was significantly lower than that of the wt AIPL1 (KD 27 μM) (B). The unique “insert” region of the FKBP domain and its α3-helix were shown to be critical for the ability of AIPL1 to chaperone PDE6 (A) (B).FKBP51 has been recently shown to preferentially and asymmetrically interact with the closed HSP90 conformation with a 1:2 stoichiometry . The asy1–316) A. Using D 27 μM) B. The unone PDE6 A 38). F. F1–316)PDE6 (A) B.AMPPNP/AIPL1 complex, the reconstituted proteins were cross-linked with disuccinimidyl suberate (DSS) or glutaraldehyde. The main product of the HSP90 dimer self-crosslinking migrated on an SDS-PAGE gel at ∼210 kDa, apparently because of extensive modification of the protein (A), and crosslinking of such a dimer to HSP90 dimer would also yield a minor 268 kDa product.To probe the stoichiometry of the HSP90 protein  and S3.  protein  and S3 a protein . These m protein A, and crA) , indicating that AIPL1 induces folding of at least a fraction of the enzyme to its active form.To investigate the role of HSP90 in PDE6 maturation, we first tested the effects of HSP90 inhibitor DMAG in a heterologous expression system based on cotransfection of HEK293T cells with PDE6C, Pγ, and AIPL1 . In thisFig. 2A) . The proA and B). The DMAG treatment also decreased proteolytic stability of AIPL1 in transfected HEK293T cells, as evidenced by the appearance of the ∼30 kDa proteolytic fragment (B). Thus, AIPL1 is proteolytically most stable when HSP90 is uninhibited, thereby supporting the interaction of AIPL1 with endogenous HSP90 in transfected HEK293T cells.We used low concentrations of DMAG, 35 and 100 nM, that had no effect on the growth and viability of transfected HEK293T cells. 17-dimethylaminoethylamino-17-demethoxygeldanamycin potently inhibited the expression of PDE6C activity in a dose-dependent manner, whereas the protein levels of the enzyme were not significantly altered , A and Bfragment B. Thus, AMPPNP (1–316 first. This mutant completely failed to induce cGMP hydrolysis in HEK293 cells cotransfected with PDE6C and Pγ (A). Counterparts of K265 in different TPR-domain containing proteins make critical contacts with the C-terminal signature motif MEEVD of HSP90 (A). The chaperone activities of AIPL1-K265A and K265W toward PDE6C in the presence of Pγ in HEK293T cells were markedly reduced (A) . Both mutations, K265A and K265W, also disrupted the binding of AIPL1 to HSP90AMPPNP (B). Consistent with the stabilizing effect of HSP90 binding on AIPL1, a minor ∼30 kDa proteolytic fragment was observed for all of the three tested mutants, AIPL11–316, K265A, and K265W . The levels of the PDE6A and PDE6B proteins were similar to those in control mice (B). The dual prenylation of PDE6 provides for its photoreceptor disc membrane attachment under isotonic conditions .Given the apparent 2:1 stoichiometry of the HSP90sylation . We exam animals  and from animals A. The lerol mice B. The dunditions , 41. Isomembrane C and S7.g cilium D.Figure C857S/C857S and control C57Bl retina homogenates were comparable (A). Likewise, the a- and b-wave ERG responses of the mutant and control mice were comparable (B), indicating overall normal phototransduction in photoreceptor cells and proper signaling to downstream rod bipolar cells.The functional properties of the mutant PDE6 and its ability to support phototransduction were assessed in PDE6 activity assays and by recording scotopic ERG responses. Basal PDE6 activity was assessed in retina homogenates obtained from dark-adapted mice, and the maximal PDE6 activity was measured after treatment with trypsin to selectively remove the inhibitory Pγ subunit . Both thmparable A. Likewimparable B, indicaA). Similarly to the wt PDE6C, maturation of PDE6C-C855S was sensitive to the HSP90 inhibitor DMAG (A).Normal maturation of PDE6 lacking farnesyl modification in mouse rods suggested that either a single prenyl moiety is sufficient for the HSP90/AIPL1 complex to bind to and induce active conformation of the enzyme or that the chaperone complex can produce such a conformational change in the absence of the client prenylation. To explore the later possibility, we coexpressed the PDE6C mutant C855S lacking the prenylation site with AIPL1 and Pγ in HEK293T cells. Remarkably, the expressed activity level of PDE6C-C855S was even moderately higher than that for PDE6C controls A. Similator DMAG A.Figure A).About 30 C-terminal residues linking the core catalytic domain of PDE6 and the prenylation site are not strongly conserved, and the C-termini are flexible and/or unstructured as indicated by the cryo-EM structure of rod PDE6 . CoexpreFig. 5B). Thus, despite the reduced stability of the ternary complex with unprenylated PDE6, the HSP90/AIPL1 complex was an effective chaperone for such a client.The lack of prenylation or the C-terminal truncation of PDE6 also resulted in the appearance of the 30 kDa AIPL1 fragment in the lysates of cotransfected cells, and the abundance of this fragment was markedly elevated in the presence of DMAG B. Thus, A). Mutant AIPL1 proteins, I61D, L100D, and L104D, completely failed to chaperone both PDE6C and PDE6C-C855S , suggesting destabilization of the hydrophobic FKBP core and/or the dynamics of the “insert” region. The chaperone activity of I61Q was markedly impaired compared to that of AIPL1 . Steric occlusion of the prenyl-binding site was pursued next by substituting I61 and I151 with a bulky Phe residue. The ability of the I61F/I151F mutant to interact with prenyl moiety was examined in the fluorescence binding assay using farnesyl-Cys-AMCA as a probe. This assay revealed a severe impairment of prenyl binding by the mutant AIPL1 (A and B). Unexpectedly, I61F/I151F was a fully competent cochaperone of both PDE6C and PDE6C-C855S in the heterologous expression system (C).The ability of the HSP90/AIPL1 complex to chaperone PDE6 with a single prenyl lipid or lacking the prenylation modifications entirely raised a question of whether prenyl binding by AIPL1 is required for proper maturation of the dually prenylated enzyme. To address this question, we generated AIPL1 mutations designed to impair prenyl binding. Residue I61 from the core AIPL1-FKBP is located at the opening of the hydrophobic prenyl-binding pocket, whereas L100 and L104 are within the FKBP “insert” region that acts as a lid over the prenyl-binding site . IndividFig. S8nt AIPL1 , A and Bn system C.Figure AMPPNP apparently reflects an additional interaction involving the AIPL1-TPR helix 7 extension and the CTD of HSP90AMPPNP. Human AIPL1, compared to the mouse isoform, contains a proline-rich region located C-terminally to the TPR helix 7 extension. This region is predicted to be unstructured , for expression as an HA-tagged protein in cultured HEK293T cells. Mutations K265A, K265W, I61D, L100D, L104D, I61Q, & double I61F/I151F were introduced into AIPL1 using the standard QuikChange site-directed mutagenesis protocol.The DNA sequence encoding the AIPL1-316 (aa 1–316) was PCR-amplified from pCDNA3.1 vector harboring AIPL1 gene  and clonhttps://nebasechanger.neb.com/). PDE6C-C855S was introduced using the standard QuikChange site-directed mutagenesis protocol. A DNA sequence encoding the full-length human HSP90β (residues 1–724) was PCR-amplified from cDNA isolated from HEK293T cells and cloned into the pET15b vector using Nco1-Nde1 sites. To obtain the Avi-tagged HSP90β, HSP90β was cloned into a modified pET21a vector with the N-terminal His6-tag followed by the Avi tag and TEV cleavage site  E. coli cells and purified over Ni-NTA resin (EMD Millipore), followed by ion-exchange chromatography on a Mono Q5 column (Bio-Rad), as previously described (The full-length mouse AIPL1 and mutant AIPL1 proteins AIPL1-Δα3 (Δ111–132), AIPL1escribed , 47, 48.6-Avi-tagged HSP90β were grown in TB media supplemented with biotin (10 mg/liter), induced at OD600 of 0.6 with 250 μM IPTG, and further grown overnight at 16 °C. In vivo biotinylation was performed as described (6-Avi-tagged proteins were purified over Ni-NTA resin (EMD Millipore) using buffer A containing 250 mM imidazole for elution and further purified by Q sepharose  anion exchange chromatography followed by SEC on a HiLoad 16/600 Superdex 200 column  equilibrated against 50 mM Tris·HCl (pH 7.5) buffer containing 5 % glycerol, 200 mM NaCl, and 1 mM TCEP.Cells for expression of the Hisescribed . The cel1–316, K265A, and K265W. Binding studies were performed in 25 mM Tris, 200 mM NaCl, 2.5% glycerol, 1 mM TCEP, 0.5 mg/ml bovine serum albumin, pH 7.5. All steps were performed at 26 °C, with biosensors stirred into 0.2 ml of sample in each well at 1000 rpm at a data acquisition rate of 5.0 Hz. N-terminally biotinylated HSP90B protein was loaded onto SA sensors at a 50 to 75 μg/ml concentration for 60 to 90 s. The data for association and dissociation phases of the assay were collected as shown in B and C. To correct for baseline drift and nonspecific binding, reference sensors with bound biotinylated HSP90β were used in the BLI assays without additions of AIPL1 proteins. Kinetic data fitting was performed using FortéBio Data Analysis software 10.0. For each concentration of AIPL1 or mutants, dissociation rate constant (koff) values were calculated from the corresponding dissociation phases of the curves. These koff values were used to calculate the association rate constant (kon) values from the association phases for each concentration according to the equation: kon= kobserved-koff/[AIPL1 or mutant].An Octet RED96 system and SA-coated biosensors (FortéBio) were used to measure association and dissociation kinetics for N-terminally biotinylated hHSP90B with AIPL1, AIPL1-Δα3, AIPL1on and koff were calculated as means of the individual kon and koff values for all curves. The affinity constant KD was calculated as mean koff/mean kon or from fitting steady-state data to the equation for one site-specific binding using the GraphPad Prism 8 software.The average k2, and 1X Protease inhibitor cocktail . Protein concentrations were measured using Bradford Assay. The samples were treated with 0.1 mg/ml TPCK-Trypsin (Sigma) on ice for 10 min to selectively degrade Pγ, after which trypsin was inhibited with the addition of 10-fold excess of soybean trypsin inhibitor (Sigma). Cell lysates (protein concentration ∼5 mg/ml) were diluted 4 to 600 fold into 40 μl  of 20 mM Tris–HCl (pH 7.5) buffer containing 120 mM NaCl, 2 mM MgSO4, 1 mM BME, 0.1 U bacterial alkaline phosphatase, and 10 μM [3H]cGMP  (PerkinElmer) for 15 min at 37 °C. The reaction was stopped by adding AG1-X2 cation exchange resin (0.5 ml of 20% bed volume suspension). The samples were incubated for 6 min at 25 °C with occasional mixing and spun at 10,000g for 3 min. 0.25 ml of the supernatant was removed for counting in a scintillation counter.For assays of PDE6 activity in HEK293T cell lysates, the cells were cultured and maintained in DMEM containing 10% fetal bovine serum (Gibco), transfected with plasmids encoding human PDE6C (or mutants), mouse Pγ, and mouse AIPL1 (or mutants) (1 μg each) using FuGene6 (Promega) according to manufacturer’s instructions. The lysates from HEK293T cells were prepared 48 h posttransfection by sonication with two 4-s pulses in isotonic buffer (20 mM Tris–HCl (pH 7.5), 120 mM KCl, 1 mM MgCl4, and 1 mM BME. After brief centrifugation  to remove cell debris, retinal homogenates  were used to measure basal PDE6 activities with final dilutions of 1:150 to 1:200 based on protein concentration. Maximal (trypsin-activated) PDE6 activities were measured from retinal homogenates treated with trypsin (100 μ/ml) for 10 min at 25 °C. Trypsin treatment was terminated by the addition of 10X soybean trypsin inhibitor (Sigma) and incubation for 5 min at 25 °C, followed by centrifugation at 20,000g for 3 min at 4 °C. The final dilutions of trypsin-treated retinal homogenates in the assays of maximal PDE6 activity were 1:4000 to 1:6000, and the activity assays were carried out as above.For the assays of PDE6 activity in retina homogenates, for each genotype at two ages (6 weeks and 6 months old), two mouse retinas were homogenized by sonication (two 5-s pulses) in 120 μl of 20 mM Tris–HCl buffer (pH 7.5) containing 120 mM NaCl, 1 mM MgSOHSP90β and AIPL1 proteins were purified by SEC in 25 mM Hepes (pH7.5) with 200 mM NaCl, 5 % Glycerol, and 1 mM TCEP. Cross-linking reactions were initiated with the addition of DSS  or glutaraldehyde (0.03%) to HSP90β (15 μM), AIPL1 (25 μM), or a mixture of HSP90β with AIPL1 and were allowed to proceed for 15 to 30 min at 25 °C after which they were quenched with the addition of 30 mM Tris–HCl (pH 7.5) . Cross-linked proteins were resolved on 3 to 8 % NuPage-Tris Acetate gel.Mass Photometry experiments were performed on a Refeyn TwoMP (Refeyn Ltd) . No.1.5,C857S/C857S mice is described in Generation of Pde6at1 K-20 (Santa Cruz Biotechnology), anti-HA (BioLegend), and anti β-actin . Procedures for immunoblotting, retinal morphology, and immunofluorescence were performed as previously described  essentially as described previously . ElectroMice were injected with a standard mixture of ketamine/xylazine /kg body weight . Upon anesthesia, eyes were hydrated with balanced salt solution (Alcon Laboratories). To obtain retinal images, the tear film was wicked away and eyes were imaged with a Bioptigen spectral domain optical coherence tomographer  using the mouse retinal bore. The volume intensity projection was centered on the optic nerve. Scan parameters were as follows: rectangular volume scans 1.4 mm in diameter, 1000 A-scans/B-scan, 100 B-scans/volume, 1 frame/B-scan, and 1 volume. After imaging, eyes were hydrated with artificial tears and mice were provided supplemental indirect warmth for anesthesia recovery. Retinal thickness was measured from retinal images within the Bioptigen InVivoVueClinic Software four times per eye using vertical angle-locked B-scan calipers from the internal limiting membrane to the retinal pigment epithelium. The six retinal thickness measurements obtained from two eyes of each animal were averaged together to produce a single retinal thickness measurement per animal. All retinal thickness measurement values are reported as an average ± SD.Labeling of S-farnesyl-L-cysteine FC with AMCA (6-((7-amino-4-methylcoumarin-3-acetyl)amino) hexanoic acid, succinimidyl-ester) was performed as described previously . Emissiot test and one-way ANOVA.Unless otherwise indicated, measurements were taken from at least three independent experiments, and the data are shown as mean value and standard error. The data are plotted in a box and whiskers type of graph, where whiskers represent minimum and maximum, boxes represent interquartile range, line represents the median, and dots represent data points. Measurements were compared using All data are contained within the article or the Supplementary Information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


In contrast, [Nix37–Pdx7+(CO)48]6– (x = 0.69) (26–) and [HNix37–Pdx7+(CO)48]5– (x = 0.53) (35–) were obtained from the reactionsof [NBu4]2[Ni6(CO)12]with 0.9–1.0 molar equivalent of [Pd(CH3CN)4][BF4]2 in thf. After workup, 35– was extracted in acetone,whereas 26– was solublein CH3CN. The total structures of 16–, 26–, and 35– weredetermined with atomic precision by single-crystal X-ray diffraction.Their metal cores adopted cubic close packed structures and displayedboth substitutional and compositional disorder, in light of the factthat some positions could be occupied by either Ni or Pd. The redoxbehavior of these new Ni–Pd molecular alloy nanoclusters wasinvestigated by cyclic voltammetry and in situ infrared spectroelectrochemistry.All three compounds 16–, 26–, and 35– displayed several reversible redoxprocesses and behaved as electron sinks and molecular nanocapacitors.Moreover, to gain insight into the factors that affect the current–potentialprofiles, cyclic voltammograms were recorded at both Pt and glassycarbon working electrodes and electrochemical impedance spectroscopyexperiments performed for the first time on molecular carbonyl nanoclusters.The molecular nanocluster[Ni The synthesis, molecular structure, electrochemical,andinfrared spectroelectrochemical behavior of three new atomically preciseNi−Pd alloy carbonyl nanoclusters are reported, together withthe first electrochemical impedance spectroscopy investigation ofredox active carbonyl nanoclusters. This behavior is somehow relatedto the fact that the metallic core of the cluster undergoes a transitionfrom an insulator to a semiconductor and, eventually, conductor regimeas its size (nuclearity) increases. As this metal core is shieldedby an insulator layer of carbon monoxide, multivalent HNMCCs may beviewed as molecular nanocapacitors.2Veryoften, high-nuclearity metal carbonyl clusters (HNMCCs) displayextended redox activity affording reversible electron cascades.1 In particular, molecular Fe and Co carbonyl clusters have been employedas electrocatalysts for the two-electron reduction of two protonsto molecular hydrogen and for the two-electron reduction of H+ and CO2 to formate.7 The molecularnature of these clusters allows us to gain detailed structural insightinto the electrocatalytic active species and into the overall mechanismoccurring in the catalyst microenvironment.8 In this sense, the study of larger and larger molecular clusterssuch as HNMCCs represents an exciting frontier that could create abridge to contemporary nanomaterials for electronic and electrochemicalapplications.13Thiselectron-sink behavior enables the application of metal carbonylclusters in electrocatalytic processes.14 In several cases, in situ infrared spectroelectrochemistry(IR SEC) proved to be crucial in proving the existence of differentstable redox states in HNMCCs.18 This is due to the fact that often low currents andbroad peaks are observed in the current–potential profile fromcyclic voltammetry (CV) of large clusters, and in several cases, thepeaks are hardly observable. Therefore, the chemical and electrochemicalreversibility of the redox processes, as well as the number of exchangedelectrons, cannot be inferred from CV and controlled potential coulometricmeasurements.The redox behavior of HNMCCs has been investigated by electrochemicaland spectroelectrochemical methods, and recently, we reported thefirst two cases of multivalence in large Ni–Pd molecular nanoclusters.19 the potential of the working electrode(WE) is swept at a slow rate between selected potentials and a sequenceof vibrational spectra in the CO stretching region (νCO) is collected at constant time intervals. Via proper selection ofthe cyclic potential range, it is possible to observe a completelyreversible shift of the νCO bands of the complexestoward higher or lower wavenumbers as a consequence of an oxidationor a reduction, respectively, according to the scan direction. Thisapplies to both terminal (νtCO) and bridging(νbCO) carbonyl groups.When the redox chemistry of a carbonyl complexis studied by IRSEC in an optically transparent thin layer electrochemical (OTTLE)cell,tCO bands within a range of 14–20 cm–1 appears to be consistent with a one-electron stepfor HNMCCs.22 Accordingly, a double shift of ∼28 cm–1 was observed for bielectronic processes.23Multipleredox changes occur sweeping the WE potential on solutionsof multivalent HNMCCs. The complete sequence of collected IR spectracan be separated into groups each belonging to a different redox step,and the spectra assigned to the electrogenerated long-lived specieswith different charges. Moreover, the presence or absence of well-definedisosbestic points in each group of spectra indicates the relativestability of the electrogenerated species or other phenomena complicatingthe electron transfers, while a nearly uniform shift of the νCO bands, indicating structural changeswith regard to the stereochemistry of CO ligands,15 according to the more favored bridging coordination modefor CO ligands at increased negative charges on a cluster.25Two further interesting pieces of information can be gatheredfromthe IR SEC analysis: (a) the stability of the electrogenerated specieson a time scale greater than that of CV, eventually suggesting thepossibility of synthesizing and isolating such reduced or oxidizedspecies, and (b) the structural variations ensuing from the redoxprocesses that cause an appreciable modification of the IR spectra.For example, in several instances, we found that changing the clustercharge resulted in a variation in the relative intensity of terminaland bridging ν39 Electrochemistry has been extremely powerful in investigating theelectronic structure of thiolate-protected nanoclusters as well asunraveling doping  effects.42 Heterometallic HNMCCsmay be viewed as atomically precise alloy nanoclusters and may contributeto a better understanding of the effects of size and structure onmolecular clusters.44 In particular, we were interested in shedding more light on thedifferent phenomena affecting their electrochemical behavior. Herein,we report the synthesis of three new heterometallic HNMCCs, [Nix36–Pdx5+(CO)46]6– (x = 0.41)(16–), [Nix37–Pdx7+(CO)48]6– (x = 0.69) (26–), and [HNix37–Pdx7+(CO)48]5– (x = 0.53) (35–). Their total structures have beendetermined by single-crystal X-ray diffraction (SC-XRD), and theirredox behavior has been investigated by CV and in situ IR SEC. Moreover,to gain insight into the factors that affect the current–potentialprofiles, the cyclic voltammograms were recorded at both Pt and glassycarbon (GC) working electrodes and electrochemical impedance spectroscopy(EIS) experiments performed for the first time on molecular carbonylnanoclusters.From a more general perspective, atomically precise metalnanoclustersand alloy nanoclusters have attracted renewed interest in recent years.22.16(CO)12]2– with miscellaneous Pd(II) salts (14 Thereaction between [Ni6(CO)12]2– and Pd(II) salts is strongly affected by several parameters suchas (a) the [Ni6(CO)12]2– cation,(b) the ligands coordinated to Pd(II), (c) the solvent, (d) the stoichiometricratio of the reagents, (e) the rate of the addition of Pd(II) to [Ni6(CO)12]2–, and (f) whether COis removed during the reaction. By carefully controlling all of theseparameters, one can obtain several high-nuclearity Ni–Pd carbonylclusters; [Ni36Pd8(CO)48]6–,45 [Ni13Pd13(CO)34]4–,46 [Ni16Pd16(CO)40]4–,47 and [Ni26Pd20(CO)54]6–47 have alreadybeen fully characterized. More recently, we have reported the synthesisand total structural characterization of [Nix22–Pdx20+(CO)48]6– (x = 0.63), [Nix29–Pdx6+(CO)42]6– (x = 0.09), and [Nix29+Pdx6–(CO)42]6– (x = 0.27).14 [Nix22–Pdx20+(CO)48]6– (x = 0.63) has been obtained from the reactionof [NBu4]2[Ni6(CO)12]with 0.7–0.8 molar equivalent of Pd(Et2S)2Cl2 in CH2Cl2, whereas [Nix29–Pdx6+(CO)42]6– (x = 0.09) and [Nix29+Pdx6–(CO)42]6– (x = 0.27) have beenobtained from the reaction of [NEt4]2[Ni6(CO)12] with 0.6–0.7 molar equivalent ofPd(Et2S)2Cl2 in CH3CN.14Bimetallic Ni–Pdcarbonyl clusters can be conveniently obtainedfrom the redox condensation of 6– (x = 0.41) (16–), [Nix37–Pdx7+(CO)48]6– (x = 0.69) (26–), and [HNix37–Pdx7+(CO)48]5– (x = 0.53)(35–). In all of thesereactions, CO was periodically removed under reduced pressure andthe Pd(II) reagent was added in small portions.During this work, we have determined the followingconditions forthe syntheses of the new species [Ni16– has been obtainedby reacting [NMe3(CH2Ph)]2[Ni6(CO)12] with 0.8 molar equivalent of [Pd(CH3CN)4][BF4]2 in tetrahydrofuran(thf), and the final product extracted in CH3CN after workup.(a) 26– and 35– have been obtained from the reactionsof [NBu4]2[Ni6(CO)12]with 0.9–1.0 molar equivalent of [Pd(CH3CN)4][BF4]2 in thf. After workup, 35– was extracted in acetone,whereas 26– was solublein CH3CN. 26– and 35– can be interconvertedby means of acid–base reactions.(b) 16–, 26–, and 35– havebeen structurally characterized by SC-XRD as their [NMe4]2[NMe3CH2Ph]4[1]·3CH3CN·solv, [NBu4]6[2]·6CH3CN, and [NBu4]5[3]·2CH3COCH3·solv salts, respectively. Their molecular structuresare discussed below.35– has been indirectly inferredfrom the factthat it is quantitatively deprotonated to the related hexa-anion bystrong bases such as NaOH, and the reaction is completely reversedafter the addition of strong acids such as HBF4·Et2O, as evidenced by IR spectroscopy. Similarly, 26– can be protonated to the relatedpenta-anion with HBF4·Et2O, and the reactionis reversed using NaOH. The fact that these penta/hexa-anion couplesare reversibly interconverted by acid–base reactions suggeststhat these are protonation/deprotonation and not redox reactions.Moreover, 26– and 35– have very similar metalcompositions . Thus, the fact that they displaydifferent electrochemical properties 48]6– and [Ni35Pt9(CO)48]6– clusters.45 Theproblem of detecting hydrides in HNMCCs has been previously discussedin the literature, and it has been shown that above a nuclearity of20–25 metal atoms, the hydride resonances in the 1H nuclear magnetic resonance (NMR) spectra become very broad andeventually HNMCCs become NMR silent.48 In agreementwith this trend, it has not been possible to directly detect the hydrideligand of 35– by 1H NMR spectroscopy under any experimental condition employed. Thus, we can onlyindirectly infer its hydride nature on the basis of the considerationsreported above.The presence of one hydride ligand on ties see 2.2 is be16– may be derived from a cubic close packed(ccp) M40 framework composed of four ABCAlayers consistingof 3, 7, 12, and 18 atoms, respectively 46]6–.50 The main differencebetween 16– and [Ni37Pt4(CO)46]6– is thatin the latter there is a perfect segregation of Ni and Pt, all fourPt atoms being fully interstitial and all Ni atoms present on thesurface of the cluster. Conversely, in the case of 16–, beside the fully interstitial Pd4 tetrahedron, there is an additional Pd atom disordered overthree symmetry-related (by a 3-fold axis) positions. These three positionsare located at the center of the three distorted (111) faces of themetal cage of the cluster. A free refinement of the occupancy factorof the independent site resulted in 0.526(18) Ni and 0.474(18) Pd.Because of the 3-fold symmetry, this corresponds to 1.59 Ni and 1.41Pd atoms per cluster unit. Thus, 16– displays both substitutional and compositional disorder, beingactually a mixture of [Ni36Pd5(CO)46]6– (59%) and [Ni35Pd6(CO)46]6– (41%). It must be mentioned that thesurface Pd atoms are at the center of almost flat centered hexagonalNi6Pd faces, displaying high M–M and low M–COconnectivities, as previously found in other Ni–Pd carbonylclusters . The clusterdisplays 508 cluster valence electrons  that correspondto 6n + 8  cluster valence orbitals (CVOs) assuming that each orbitalis filled with two electrons.2 This electroncount is in keeping with other Ni–Pd carbonyl clusters. Thus,[Ni13Pd13(CO)34]4–,46 [Ni16Pd16(CO)40]4–,47 [Nix29–Pdx6+(CO)42]6– (x = 0.09),and [Nix29+Pdx6–(CO)42]6– (x =0.27)14 display 6n + 10CVOs; [Ni26Pd20(CO)54]6– displays 6n + 11 CVOs,47 [Nix22–Pdx20+(CO)48]6– (x =0.63) 6n + 9 CVOs,14 and[Ni36Pd8(CO)48]6– 6n + 7 CVOs.45 It mustbe mentioned that this is just an empirical rule for electron bookkeepingin HNMCCs, where the CVE number merely results from the sum of thevalence electrons of each metal , the electrons arising from the ligands (two for CO), andthe total charge of the cluster.2 The numberof filled CVOs is obtained by dividing the CVE number by 2, thus assumingtwo paired electrons per orbital (diamagnetic cluster). The CVE numbercan be determined exactly, once the cluster formula is known. In contrast,the real CVO number may be greater than the calculated one, in thecase of paramagnetic clusters. At the moment, there is no generalrule for predicting a priori the CVE and CVO numbersfor HNMCCs.5 As mentioned above, it has been empirically observed that for allof the HNMCCs characterized to date, the calculated CVE number correspondsto 6n + x CVOs  when x canassume all values between 2 and 24. Even if the empirical parameter x is within a wide range, it displays similar values forrelated clusters. Thus, for Ni–Pd carbonyl clusters, x is between 7 and 11,47 whereas it can reach higher values in the case of HNMCCs containinginterstitial main group atoms such as phosphides and carbides.52The metal coreof 26– and 35– . The related[Ni36Pd8(CO)48]6– anion has been previously reported.45 Its metal framework consists of a Ni36Pd8 ν3 octahedron  encapsulatinga Pd6 octahedron. The two additional Pd atoms on the surfaceare disordered, showing a preference for the central position of theeight (111) faces. The 26– and 35– anions reportedherein display rather similar structures, and in addition to Ni/Pdsubstitutional disorder, they show compositional disorder. Thus, 26– is actually a mixture of[Ni37Pd7(CO)48]6– (31%) and [Ni36Pd8(CO)48]6– (69%), whereas 35– consistsof [HNi37Pd7(CO)48]5– (47%) and [HNi36Pd8(CO)48]5– (53%). The overall Ni/Pd distribution in both clustersis very similar to that previously observed for [Ni36Pd8(CO)48]6–. Thus, the six fullyinterstitial positions are occupied by Pd atoms, and the additionalPd atoms are disordered at the center of the eight (111) hexagonalfaces. Some minor differences in the occupancy factors of these sitesare observed for the different species. In addition, in the case of 35–, two independent moleculesare present within the unit cell, displaying some significant differencesin their compositions. Thus, the first independent molecule consistsof [HNi37Pd7(CO)48]5– (26%) and [HNi36Pd8(CO)48]5– (74%), whereas the second site contains [HNi37Pd7(CO)48]5– (68%)and [HNi36Pd8(CO)48]5– (32%).The presence of substitutional and compositional disorderin Ni–Pdcarbonyl clusters is further exemplified by the structures of 26– and 35– contain168M–M bonds and are completed by 48 CO ligands . They both display 542 CVEs and 271 CVOsas previously found for [Ni36Pd8(CO)48]6– [36 × 10 (Ni) + 8 × 10 (Pd) + 48× 2 (CO) + 6 (charge)].The metal cores of 2.22.2.116– in a CH3CN/[NBu4][PF6] solution,although not so well resolved as in the case of mononuclear complexes,is not so poorly defined as often found with other multivalent HNMCCs. Two otherpartially overlapping processes are detected at more cathodic potentials(−1.54 and −1.79 V), and another not very resolved peakis observed at −0.46 V ,while the process at −1.79 V is accompanied by an extensivedecomposition of the electrogenerated species, as indicated by theappearance and fast increase in the intensity of the band at 2127cm–1 due to dissolved CO.The CV profile of t HNMCCs4.14−18 T −0.46 V 3. On the16–  indicates that the peak at −0.46V corresponds to an oxidation. However, before the complete oxidationof 16–, a weak shoulderat 2043 cm–1 due to Ni(CO)4 indicatesa relatively slow fragmentation of the metallic core of the electrogenerated[Nix36–Pdx5+(CO)46]5– (15–). The starting spectrum of 16– was almost completely restoredin the reverse reduction back scan.A shift at higher frequencies of the two IR bands of The IR SEC study allowedus to identify five redox couples between−0.24 and −1.80 V (vs the Ag pseudoreference electrode).One oxidation step produced a cluster of limited stability; threereductions, chemically reversible in the time scale of the IR SECexperiment, demonstrated that the core of the cluster is stable withup to three additional electrons, while the addition of a fourth electroncaused a relatively fast CO evolution.x36–Pdx5+(CO)46]n− (n = 5–9)(1n–)were selected  and bridging (νbCO) carbonylgroups for each species are listed in bCO bands compared to the νtCO bands was observed during the consecutive reductionsteps. This pointed to some structural changes regarding the stereochemistryof CO ligands for species with increased negative charge and may foreseea slowdown of the electron transfers that is a decrease in the electrochemicalreversibility for the related redox steps.The IR spectra of thefive oxidation states of [PF6] solutionat different Pt electrodes were always less resolved than those atthe GC WE. Five processes, all possessing features of chemical reversibilityon the CV time scale, are observed between −0.3 and −2.1V  at the GC WE  without any decomposition of the electrogenerated species.Indeed, the IR spectrum of the starting cluster was restored whenthe WE potential was returned to the initial value. From the analysisof the complete sequence of the recorded IR spectra, it has been possibleto identify five redox states of 35–. In particular, during the slow sweep of the WE potentialfrom −0.21 to −1.60 V, the νCO bandsof 35– (2002 and 1860cm–1) were shifted to lower wavenumbers and threereduction processes, completely reversible on the time scale of IRSEC, were observed . With a further decrease in the applied potential from −1.6to −2.0 V, the shift of the νCO bands at lowerwavenumbers was accompanied by a clear variation of the relative intensityof the terminal and bridging νCO bands in favor ofthe bridging ones . This isin agreement with the fact that with an increase in the negative chargeof a carbonyl cluster, bridging CO ligands are more favored than terminalones.25 A similar change in the stereochemistryof the CO ligands was previously observed for other clusters. Thischange was related to the electrochemical quasi-reversibility of theredox processes, which retained the chemical reversibility becausea spectrum that could be superimposed with that of the starting clusterwas obtained when the WE potential was returned to the initial value.23 In the case of 35–, instead, during the back oxidationscan up to −0.21 V, the shift of the νCO bandsat the initial values was observed, but not equally restored was theirintensity ratio.When the redox chemistry of Figure S12).With a further decrease in the WE potentialfrom −2.0 to−2.2 V, CO release in solution and other modifications of theIR spectra suggested incipient decomposition of the multireduced cluster4 starting before the complete oxidation of the initial clusterindicated the limited stability of the oxidized species , and the spectrum of 35– was not completely restored inthe reverse reduction back scan .When the WE potentialwas increased from −0.21 to 0.1 V(vs the Ag pseudoreference electrode), an oxidation of the clusterwas indicated by the upshift of the νCO regionof thefive reversible redox states of cluster [HNix37–Pdx7+(CO)48]n− (n = 4–8)(3n–)are shown in tCO and νbCO stretching frequencies for each species are listedin tCO bands (∼15cm–1), all of the processes were considered monoelectronicand the charge of each species was consequently assigned.The vibrational spectra in the ν2.2.326– at GC and Pt electrodesare shown in 35– , itseemed that the two species were not the same cluster with differentoxidation states, thus supporting the presence of a hydride atom in 35–. Moreover, we excludedthe possibility that the impurities present in both samples couldbe attributed to protonation–deprotonation equilibria oftenobserved for similar Ni–Pt or Ni–Pd clusters.53The cyclic voltammogramsof 6(CO)12]2– were found in the sample of 26– used for the CV and IRSEC investigations. Such an impurityis not observed in the CV of 6(CO)12]2– is evident in the IR SEC experiments. 3CN solution of 26– when the WE potential isswept between −0.3 and −1.8 V (vs the Ag pseudoreferenceelectrode). While a downshift of νCO bands with adecrease in the potentials is clearly evident in the bridging CO region,the overlap of νtCO bands belonging to 26– (1990 and 1953 cm–1) and [Ni6(CO)12]2–  made it difficult to detectin this IR region the real shift induced by the redox processes occurringfrom −0.3 to −1.8 V (vs the Ag pseudoreference electrode).On the basis of the consecutive shift of the νbCO bands of 26– from 1865 to 1853, 1840, 1826, and 1812 cm–1,the IR spectra shown in sequence in 6(CO)12]2– interferingIR bands, which remained unchanged in the applied potential window,we calculated the differential absorbance spectra using as a referencespectrum the first one in each group. The four IR spectral changesobserved in the potential range from −0.3 to −1.8 Vare shown in Figures S16–S19. Whenthe WE potential was increased from −0.6 to −0.3 V,an upshift of both νCO bands of 26– pointed to an oxidation process, whilewith a decrease in the potential from −0.6 to −1.8 V,three consecutive downshifts of both IR absorptions of 26– were attributed to three reductionprocesses. All of the redox steps were completely chemically reversible,and the potential could be cycled between −0.3 and −1.8V without decomposition of the electrogenerated species. Indeed, theIR spectrum of the starting 26– was obtained when the WE potential was returned to the initialvalue . Instead, with an increasein the potential above −0.3 V or a decrease to values morenegative than −1.8 V, relatively fast decomposition reactionswere observed.Small amounts of 4–.20 The behavior of Rct at the E° potential of electron transfer isdirectly related to the kinetics of the electrochemical reaction  was performed toverify the similar dimensions of the electrode electroactive area22]4– recorded at a GC and at aPt WE are shown in Figure S21 and wereused to determine the E° values for EIS measurements.As shown in Z| and phase shift angle Φvs the logarithm of the frequency) plots of the impedance spectrarecorded at the E° values of successive redoxpeaks display a negligible difference in the profiles when Pt andGC electrodes are compared for the peaks at 0.06 and −0.18V. Apparently, the corresponding calculated Rct values, obtained by fitting the experimental data with theRandles circuit using the integrated tool of PStrace version 5.8,are similar (∼1 kΩ) and indicate relatively fast electrontransfers for both electrodes. On the contrary, the peaks at −1.10and −1.25 V show a higher impedance value for the Pt electrodes(Rct ∼ 5 kΩ), suggestingslower kinetics for such electrodes, as it was supposed from lowerand broader current peaks in CV .The CV responses between0.2 and −1.6 V of a CH16–, 35–, and 26–. TheEIS behaviors of 16–, 35–, and 26– at Pt and GC electrodes are comparedin A similar approach was used to study 16– exhibitedthe same behavior on both Pt and GC electrodes for all of the examinedelectron transfers, featuring relatively fast kinetics, with Rct values without significant differences betweenvoltage peaks.As shown by the plotsof 35– at the potentials of the five consecutivereduction stepson different WE materials afforded results similar to those of [Pt19(CO)22]4–. In particular, for 35– the EIS spectra of peaksat the more negative potentials could not be recorded with a Pt WE,because in the CV analysis very low currents with Pt WE made the peakshardly observable, while high Rct valueswere obtained for the peaks at −0.520 and −0.890 V , confirming slow kinetics toward thatelectrode material. We found that subsequent reductions lead to slowerkinetic behavior for 35– also on a GC WE. This can be explained by supposing important structuralmodification of the cluster as the number of added electrons increases.An increase in the metal–metal bond distances as the negativecharge of the cluster increases is expected for multivalent carbonylcompounds.55 Indeed, density functionaltheory-optimized geometries of the multivalent family of clusters[Rh12E(CO)27]n–  indicated elongation of the Rh–Rhand Rh–E bond distances for multireduced species.56The EIS measurements for 26– when comparing Pt and GCelectrodes. GC EISspectra showed fast and comparable kinetics for all of the peaks investigated.Quite the opposite results were obtained for Pt electrodes where high Rct values (15 and 42 kΩ) were calculatedfrom EIS measurements at the potentials of the only two peaks visiblefrom CV measurements .Similar results were also obtained for 316–, 26–, and 35– as well as the previouslyreported [Ni36Pd8(CO)48]6–45 are structurally related to Ni–Ptclusters [Ni37Pt4(CO)46]6–, [Ni38Pt6(CO)48]6–, and [Ni35Pt9(CO)48]6–.53 This is notso common, because more often Ni–Pd and Ni–Pt HNMCCsdisplay rather different structures. The comparison of these few examplesof structurally related Ni–Pd and Ni–Pt clusters isquite interesting. The overall structures of their metal cores andthe stereochemistry of the CO ligands are almost identical. With regardto the Ni–Pd and Ni–Pt distributions, both Pd and Pttend to completely occupy the fully interstitial positions, even iffor different reasons. Indeed, in the case of Ni–Pd carbonylclusters, Pd tends to minimize Pd–CO contacts, because Pd–CObonds are considerably weaker than Ni–CO ones.14 Conversely, in the case of Ni–Pt clusters, Pt prefersinterstitial positions to maximize Pt–Pt and Pt–Ni bonds,because Pt forms very strong metal–metal bonds. Some Ni/Pddisorder is present on the surface of Ni–Pd clusters, usuallyconcerning positions with low M–CO connectivity. This surfacedisorder is more limited in Ni–Pt clusters. These results indicatethat the total structures of molecular alloy M–M′ nanoclusterscomprise a subtle balance of the different M–ligand, M′–ligand,M–M, M–M′, and M′–M′ bondingenergies.Three new atomically preciseNi–Pd alloy carbonyl nanoclustershave been synthesized, and their total (molecular) structures determinedby SC-XRD. The new Ni–Pd HNMCCs 16–, 26–, and 35–), aswell the Ni–Pdalloy nanoclusters described in our previous paper,14 display several chemically and electrochemically reversibleone-electron redox processes, as clearly demonstrated by joint CVand IR SEC experiments. In this sense, they are multivalent and behaveas electron sinks and molecular nanocapacitors. Several additionalpieces of information can be obtained from detailed IR SEC analyses:(a) the appearance of chemically reversible redox processes even whenthe peaks in their CV profiles are very weak, (b) the number of exchangedelectrons, based on the νCO shift, (c) stereochemicalrearrangements of the CO ligands accompanying the reversible additionand/or removal of electrons (by comparing the relative intensity ofνtCO vs that of νbCO), and (d) the occurrence of irreversible processes and decompositionreactions. Point (a) has been somehow debated, because it was notvery clear why in some cases very low currents were detected in CVexperiments leading to almost featureless i/V profiles, whereas for the same species very clear νCO shifts were present in the IR SEC experiments suggestingwell-defined reversible redox processes. To shed light on this point,we performed CV experiments with different WE materials and, for thefirst time, EIS analyses of 16–, 26–, and 35–, as well as redox active[Pt19(CO)22]4–, whose electrochemistrywas reported in detail previously.20 Allof these experiments demonstrated that the kinetics of the electronexchange at the electrode surface interfaces is a critical point.For some cluster/WE material combinations, this process is very slow,leading to low currents and poorly resolved peaks in the CV profiles.In these cases, better insight into the electrochemical behavior ofthe clusters can be gained by IR SEC experiments, which seem to sufferfewer of these problems.The three HNMCCs reported herein  can be obtained by SC-XRD, whereas electrochemicaland spectroelectrochemical experiments offer detailed informationabout the electronic status of their metal cores and structural, spectroscopic,and chemical information as the number of electrons is varied.44.14]2[Ni6(CO)12], [NMe3CH2Ph]2[Ni6(CO)12],57 and [Pd(CH3CN)4][BF4]2,58 which were prepared according to the literature. Analyses of C,H, and N were performed with a Thermo Quest Flash EA 1112NC instrument.Analyses of Ni and Pd were performed by microwave plasma-atomic emissionspectrometry on an Agilent 4210 MP-AES instrument. IR spectra wererecorded on a PerkinElmer Spectrum One interferometer in CaF2 cells. Structural drawings were determined with SCHAKAL99.59All reactionsand sample manipulations were carried out using standard Schlenk techniquesunder nitrogen and in dried solvents. All of the reagents were commercialproducts (Aldrich) of the highest purity available and used as received,except [NBuCaution: CO and Ni(CO)4 may be generated during manipulation of these compounds.All of the operations must be carried out under a well-ventilatedfume hood.4.23CN)4][BF4]2 was added as a solid in small portions to a solution of [NMe3CH2Ph]2[Ni6(CO)12]  in thf (80 mL) over a period of 6 h. The resulting mixturewas stirred at room temperature for 24 h, and then the solvent removed in vacuo. The residue was washed with H2O (3× 20 mL) and extracted with acetone (20 mL). A saturated solutionof [NMe4]Cl in H2O (50 mL) was added to completethe precipitation of the compound. The solid was recovered by filtration,washed with H2O (3 × 20 mL), toluene (3 × 20mL), and thf (20 mL), and extracted with CH3CN (20 mL).Crystals of [NMe4]2[NMe3CH2Ph]4[1]·3CH3CN·solvsuitable for X-ray analyses were obtained by layering n-hexane (2 mL) and diisopropyl ether (40 mL) on the CH3CN solution .Anal. Calcd for C100H97N9Ni35.58O46Pd5.41 (4826.21): C, 25.03; H, 2.04; N,2.63; Ni, 43.00; Pd, 11.95. Found: C, 25.34; H, 2.29; N, 2.31; Ni,43.28; Pd, 12.15. IR  νCO: 2005(vs), 1858(s) cm–1.[Pd(CH4.33CN)4][BF4]2  was added as a solid in small portions to a solutionof [NBu4]2[Ni6(CO)12] in thf (30 mL) over a period of 6 h. The resultingmixture was stirred at room temperature for 24 h, and then the solventremoved in vacuo. The residue was washed with H2O (3 × 20 mL), toluene (3 × 20 mL), and thf (20mL) and extracted with CH3CN (20 mL). Crystals of [NBu4]6[2]·6CH3CN suitablefor X-ray analyses were obtained by layering n-hexane(2 mL) and diisopropyl ether (40 mL) on the CH3CN solution. Anal. Calcd forC156H234N12Ni36.31O48Pd7.69 (5995.51): C, 31.40; H, 3.96; N, 2.82;Ni, 35.29; Pd, 13.66. Found: C, 31.16; H, 4.12; N, 2.65; Ni, 34.98;Pd, 13.89. IR  νCO: 1983(vs),1838(s) cm–1.[Pd(CH4.43CN)4][BF4]2  was added as a solid in smallportions to a solution of [NBu4]2[Ni6(CO)12]  in thf (30 mL) over a periodof 6 h. The resulting mixture was stirred at room temperature for24 h, and then the solvent removed in vacuo. Theresidue was washed with H2O (3 × 20 mL), toluene (3× 20 mL), and thf (20 mL) and extracted with acetone (20 mL).Crystals of [NBu4]5[3]·2CH3COCH3·solv suitable for X-ray analyses wereobtained by layering n-hexane (40 mL) on the acetonesolution . Anal.Calcd for C134H192N5Ni36.47O50Pd7.53 (5615.50): C, 28.81; H, 3.47; N,1.25; Ni, 37.85; Pd, 14.29. Found: C, 28.64; H, 3.26; N, 1.51; Ni,37.98; Pd, 14.02. IR  νCO: 2004(vs),1972(sh), 1873(s), 1854(sh) cm–1. IR  νCO: 2013(vs), 1875(s) cm–1.IR  νCO: 2006(vs), 1867(s)cm–1.[PF6].62 The GC electrode was polished, prior to measurements, accordingto the following procedure: manual rubbing with a 0.3 μm Al2O3 slurry in water (eDAQ) for 2 min, sonicationin ultrapure water for 10 min, manual rubbing with a 0.05 μmAl2O3 slurry in water (eDAQ) for 2 min, andsonication in ultrapure water for 10 min. After being polished, theelectrodes were rinsed with acetone and air-dried.A platinum disk (7.39× 10Edc the E°′ of a reversible electrochemicalreaction obtained from the voltammetric experiments. Eac was set to 0.005 V, and the frequency was scanned between10000 and 1 Hz. All of the electrochemical experiments were performedusing a Palmsens 4 potentiostat.EIS spectrawere recorded using as