In this review, we highlight some of the key findings to date in the discovery of PROTACs targeting HDACs by HDAC class and HDAC isoenzyme, current gaps in PROTACs to target HDACs and future outlooks.Over the past three decades, we have witnessed the progression of small molecule chemical probes designed to inhibit the catalytic active site of histone deacetylase (HDAC) enzymes into FDA approved drugs. However, it is only in the past five years we have witnessed the emergence of proteolysis targeting chimeras (PROTACs) capable of promoting the proteasome mediated degradation of HDACs. This is a field still in its infancy, however given the current progress of PROTACs in clinical trials and the fact that FDA approved HDAC drugs are already in the clinic, there is significant potential in developing PROTACs to target HDACs as therapeutics. Beyond therapeutics, PROTACs also serve important applications as chemical probes to interrogate fundamental biology related to HDACs  A review on current proteolysis targeting chimeras (PROTACs) as chemical probes for histone deacetylase (HDAC) enzymes. N-ε-acetyl-l-lysine amino acids in histone proteins. The reverse reaction, histone acetylation, can also be carried out by Histone Acetyltransferases (HATs). Histone acetylation and histone deacetylation play important roles in influencing chromatin structure and gene transcription.1 For example, histones with non-acetylated lysine residues encompass positive charges and are tightly associated with negatively charged DNA, termed heterochromatin.2 Alternatively, histones with acetylated lysine residues, with no charge on the lysine residue, are less tightly associated with DNA and are less compact in histone DNA packing, termed euchromatin.2 Additionally, the acetylated lysine residues on histones also serve as important recruitment sites for bromodomain-containing proteins that can further recruit HATs or chromatin remodelers capable of altering DNA accessibility to transcription factors.3 Hence, the fine balance between HDAC and HAT activities in the cell plays an important role in the regulation of gene expression, and such post-translational modifications are considered important epigenetic markers.4Eighteen histone deacetylase (HDAC) enzymes exist in humans, primarily characterised for their catalysis in the hydrolysis of acetyl groups from 5–8 p53, STAT3 and AR are important transcription factors whereby their overexpression or mutation can be associated with many cancers.9–11 While α-tubulin, another important cancer drug target, is a central component of the cytoskeleton and important for cell division.12Despite their role in histone deacetylation, the name Histone Deacetylase can perhaps be deceptive as the substrate specificity of HDACs can expand beyond histone proteins to non-histones such as p53, signal transducer and activator of transcription 3 (STAT3), the androgen receptor (AR) and α-tubulin as a few select examples.13 Of the eighteen HDAC enzymes in humans, eleven perform catalysis utilising a divalent Zn2+ ion in the HDAC active site, while the remaining seven are NAD+-dependent and termed Sirtuins (SIRTs).14 HDACs can be further subdivided into classes based on their sequence similarity to yeast HDACs Hda1 and Rpd3. The eleven-zinc dependent HDACs encompass class I , class II  and class IV (HDAC11), and the seven SIRTs encompass class III (SIRT1–SIRT7). Class II can be further subdivided into class IIa  and class IIb (HDAC6 and HDAC10). These isoforms differ in their sub-cellular locations, catalytic activities and substrate specificities. For example, HDAC1, HDAC2 and HDAC3 are predominantly localised in the nucleus while the majority of other zinc-dependent HDACs can be found in either the nucleus or cytoplasm.15Higher eukaryotic organisms typically contain more HDAC isoforms, the common fruit fly only encodes 5 HDAC genes in comparison to the 18 HDACs in humans.16 In comparison, since the approval of the first rationally designed kinase inhibitor imatinib in 2001, over 74 small molecule inhibitors targeting kinases gained FDA approved by the end of 2021.17 One hurdle to the potential progression of new HDAC inhibitors is perhaps the toxicities that have been associated with current FDA-approved HDAC drugs. Side effects experienced by patients taking vorinostat include fatigue, gastrointestinal upset, thrombocytopenia and QT interval prolongation,18 more akin to a non-selective cytotoxic agent than a targeted cancer therapeutic. Many have hypothesised that these side effects are linked to the pan-HDAC inhibition of all eleven zinc-dependent HDAC enzymes by inhibitors such as vorinostat.16,19–22 It could be envisaged the global change in the acetylome caused by pan-HDAC inhibition could be leading to these undesired side effects. However, it is also important to note that the hydroxamic acid functional group, incorporated in three of four FDA approved HDAC targeting drugs, have also been associated with mutagenicity.23Given the importance of HDACs in gene transcription it is perhaps no surprise that a number of HDAC inhibitors have gained FDA approval . Vorinos16,19–22 The literature on small molecule HDAC inhibitors is vast and beyond the scope of this review. We recommend the review by Ho et al. for a comprehensive account of HDAC inhibitor design and development over the past thirty years.24 At the time of writing this review 135 clinical trials have been reported as active or recruiting in clinicaltrials.gov related to the search term ‘Histone Deacetylase’.25 Many of these trials are combination therapies with FDA approved HDAC inhibitors, but also contain new HDAC inhibitors, and hybrid dual functionalised HDAC inhibitors designed to target HDACs and other protein drug targets including kinases.26 Intriguingly, current HDAC inhibitors targeting a singular HDAC isoform in clinical trials are mainly represented by HDAC6.Despite this there is still hope and enthusiasm that the discovery of isoform selective HDAC inhibitors will lead to enhanced therapeutic efficacies with reduced side effects for specific diseases related to certain HDAC isoforms.in vivo in seven differing corepressor complexes adding an additional layer of complexity.27 The differing corepressor complexes each have distinct physiological roles in the cell, and hence targeting a HDAC containing corepressor complex with selectivity may also be essential to discovering new HDAC therapeutics with greater therapeutic efficacies and reduced side effects.28 It will be truly interesting to see how the future of Proteolysis Targeting Chimeras (PROTACs) can contribute to this area.Beyond isoform selectivity, HDAC1, HDAC2 and HDAC3 exist 29–31 Chemical probes have also proved important for validating the role of specific proteins in disease and their potential as drug targets.32,33 Most chemical probes are rationally designed to inhibit protein function. However, the discovery of cell permeable molecules, not only capable of protein inhibition, but also degradation of the target protein has created a new area of research in chemical probes.34–36 Perhaps the most well-known examples of this being proteolysis targeting chimeras (PROTACs). However, given the success of this strategy other protein degradation strategies such as molecular glues, lysosome-targeting chimeras (LyTACs), autophagosome-tethering compounds (ATTECs) and antibody-based PROTACs (AbTACs) are also being actively explored.37–39 To date, the majority of protein degradation strategies targeting HDACs has been focused on the design of PROTACs which will also be the focus of this review, however Toriki et al. recently reported molecular glues targeting HDAC1 and HDAC3 for degradation.40Small molecule chemical probes that exhibit high affinity and selectivity for their intended target protein serve important applications in addressing fundamental biological questions related to the target protein.41 the formation of a stabilised and cooperative ternary complex is often highlighted as important for degradation.42 This unique mechanism of action can lead to significantly enhanced protein selectivity. For example, the bromodomain inhibitor JQ1 is a pan-BET inhibitor, however when JQ1 is incorporated into a PROTAC, this results in the selective degradation of BRD4 over BRD2 and BRD3.43 Foretinib, a pan-kinase inhibitor, was found to bind 133 kinases of the 428 kinases screened by Bondeson et al.,44 however when functionalised into PROTAC 1 (utilising the Von Hippel-Lindau E3-ligand) it retained binding to just 52 of the 133 kinases  of 210 nM for p38α. The stability of the ternary complex formed between 1, p38α and the VHL ligase complex was highlighted as more important for degradation than the binding affinity between p38α and 1. Further studies revealed that modifying the connectivity of the VHL ligand to the linker in PROTAC 2, while maintaining the same foretinib POI binding ligand, can modify the selectivity of degradation for the p38δ isoform over previously degraded p38α.45 These are just a few examples of how PROTAC-mediated degradation can alter protein selectivity, and there are many recent and comprehensive reviews available on PROTACs.46–48PROTACs designed to degrade their target protein typically contain three components: a ligand to engage the protein of interest (POI), a ligand to initiate protein degradation (most commonly an E3-ligand), and a linker covalently bonding these two ligands. PROTACs result in poly-ubiquitination of the lysine amino acids on the POI resulting in eventual degradation by the proteasome. This transfer of ubiquitin to the POI requires the formation of a protein–protein interaction between the POI and E3-ligase mediated by the PROTAC, kinases . In prot+-dependent SIRT2 published online in 2017.49 The first zinc-dependent HDAC targeting PROTAC was reported for HDAC6 in 2018.50 It is important to note PROTACs also have promise beyond chemical probes with approximately 20 PROTACs progressing to or already in clinical trials.46 The most prevalent protein targets for these PROTACs being AR, estrogen receptor (ER), Bruton's tyrosine kinase (BTK) and interleukin-1 receptor-associated kinase 4 (IRAK4). To date, as of writing, no PROTACs targeting HDACs are in clinical trials.Given such remarkable findings in modifying protein selectivity by utilising PROTAC mediated degradation over enzyme/receptor inhibition it is no surprise researchers wishing to modulate HDAC activity have been drawn to the PROTAC field. The first PROTAC targeting a Histone Deacetylase enzyme was NAD28 HDAC1 and HDAC2 share 83% amino acid sequence identity and exist in vivo interchangeably as components of six corepressor complexes , while HDAC3 exists in the SMRT/NCoR complex.28 HDAC8, unlike HDAC1-3, is not known to exist in corepressor complexes.Class I HDACs encompass HDAC1, HDAC2, HDAC3 and HDAC8. HDAC1, HDAC2 and HDAC3 are predominantly localised in the nucleus of the cell, while HDAC8 can be found in both the nucleus and cytoplasm.51 while HDAC3 has recently been shown to have a critical role in tumour development in Kras-mutant non-small cell lung cancer.52 HDAC8 has been found to be overexpressed in a number of cancers.53 To date, a number of PROTACs have been reported capable of degrading class I HDACs.54–68Of the HDAC family, HDAC1–HDAC3 are attractive drug targets given that they are nucleus-localised and hence likely the most prominent HDACs in regulating histone deacetylation and gene transcription. The selective targeting of HDAC1 and HDAC2 has been highlighted as a potential strategy for treating B-cell acute lymphoblastic leukaemia (B-ALL),et al. carried out a proteomics study investigating HDAC degradation in the presence of 48 PROTACs that varied in HDAC ligand, linker length and E3 ligand.56 In this study HDAC1, HDAC2 and HDAC9 were the least frequently identified HDAC isoforms to undergo proteasome mediated degradation of 9 of the 11 zinc-dependent HDACs on PROTAC treatment (HDAC10 and HDAC11 are in low abundance in the KELLY cell line used in the study). On the other side of the spectrum, HDAC6, HDAC8 and HDAC3 were the most frequently identified HDAC isoforms to undergo proteasome mediated degradation with the 48 PROTACs used in the study. This seems to be the challenge in generating HDAC1 and HDAC2 selective PROTACs, separating HDAC1 and HDAC2 degradation from other isoform degradation such as HDAC3 and HDAC6 degradation, the latter of which seem more susceptible to PROTAC mediated degradation, and there are other examples of this beyond the study by Xiong et al.Although PROTACs have been reported capable of degrading HDAC1 and HDAC2 achieving the selective degradation of these isoforms has proved challenging. Xiong et al. were able to synthesise PROTAC 3, which was capable of degrading HDAC1 at a concentration of 10 μM in the acute myeloid leukaemia HL-60 cell line  approach Sinatra ell line .57 Howev50 value of 0.55 μM for HDAC1 and still degraded HDAC2 at low micromolar concentrations, while 5 exhibited DC50 values of 0.91 μM and 4.19 μM respectively for HDAC1 and HDAC2.58 These PROTACs are also effective degraders of HDAC3 , however we noticed a significant observation in that these PROTACs exhibit a hook effect for HDAC3. At concentrations greater than 1 μM HDAC3 abundance starts to increase rather than decrease, while reduced HDAC1 and HDAC2 abundance was still maintained at concentrations greater than 1 μM. This effect was also time dependent and at 36 hours HDAC3 abundance started to reduce again with 4 meaning caution must be taken not only with the PROTAC concentration used but also with time-dependent degradation. PROTACs 4 and 5 were capable of compromising cell viability in HCT116 cells with single-digit micromolar IC50 values, however they were only marginally enhanced in potency compared to the benzamide class I HDAC inhibitor CI-994. In a separate study, we investigated utilising the class I HDAC inhibitor entinsotat as inspiration for PROTAC 6.59 PROTAC 6 encompassing the triazole and carbamate moiety was also a degrader of HDAC1, HDAC2 and HDAC3 in HCT116 cells, but was reduced in degradation potency (HDAC1 DC50 = 2.8 μM) compared to 4 and 5. PROTAC 6 also exhibited a similar hook effect for HDAC3 although at higher concentrations, above 2.5 μM.PROTAC 4 exhibited a DCvice versa given the high amino acid sequence similarity between HDAC1 and HDAC2. With PROTACs 4 and 5 the dose response curves for HDAC1 and HDAC2 degradation mirror one another, however we have consistently observed greater HDAC1 degradation levels over HDAC2 degradation,54,58,59 at least suggesting this may be viable. Regards targeting HDAC1 and HDAC2 for degradation, there are PROTACs reported that degrade HDAC1 and HDAC2, however not with complete selectivity and work still needs to be done to fine tune HDAC1 and HDAC2 degradation over the degradation of other HDAC isoforms.A real feat of the PROTAC technology would to be selectively degrade HDAC1 over HDAC2 or 56,58,60,61 Using a hydrazide pan HDAC1-3 inhibitor, an uncommon HDAC ligand, Xiao et al. were able to synthesise and identify a HDAC3 selective degrader, 7, incorporating an alkyl linker and a VHL ligand in the PROTAC  compared to 4 and 5 that also degrade HDAC1 and HDAC2. 11 also did not compromise cell viability in HCT116 cells to the same levels as PROTACs 4 and 5 and the pan-HDAC1-3 inhibitor CI-994.In optimisation studies of PROTACs targeting HDAC1-3 for degradation we modified the attachment of the VHL E3-ligand to the linker in PROTAC 11.The selective degradation of HDAC3 by PROTACs seems to be achievable. PROTACs that recruit the VHL E3 ligase are more likely to promote the degradation of HDAC3. However, it is also important to note that the choice of cell line can have a crucial effect on the HDAC isoform degradation outcome.in vivo.56,62 One of few studies was carried out by Xiong et al., investigating the effects of 48 PROTACs on HDAC1-3 containing corepressor complexes by proteomics.55 Reduced abundance of GPS2, NCoR1 and NCoR2, components of the HDAC3-SMRT/NCoR complex, coincided with PROTACs capable of degrading HDAC3, including PROTACs 9 and 10. The reduced abundance of these HDAC3-SMRT/NCoR corepressor complex components was much more prominent with PROTACs recruiting the VHL E3 ligase compared to PROTACs recruiting the cereblon E3 ligase (HDAC ligand dacinostat). Intriguingly, MIER1–3, components of HDAC1/2 containing corepressor complexes, were also reduced in abundance with certain PROTACs, including 9 and 10, despite no observed significant reduction in HDAC1 and HDAC2 abundance. Three PROTACs recruiting the IAP E3 ligase were capable of reducing the abundance of GSE1 and HMG20B components of the CoREST complex. The authors hypothesised that such PROTACs can engage HDAC1 and HDAC2 in these corepressor complexes but only result in the degradation of these specific complex components and not the degradation of HDAC1 and HDAC2 themselves.Very few studies have investigated the effects of HDAC targeting PROTACs on the corepressor complexes that HDAC1, HDAC2 and HDAC3 exist 62 We compared the gene expression profile of multiple components of all seven HDAC1–3 containing corepressor complexes. Although not considered differentially expressed genes, genes of the HDAC3-SMRT/NCoR complex TBL1RX1, TBL1X and NCoR1 were exclusively only upregulated in the presence of the HDAC3 selective PROTAC 11, highlighting again, similar to the results by Xiong et al., that this corepressor complex does seem to be affected as whole by PROTAC-mediated degradation.We performed RNA-seq experiments with the HDAC1–3 inhibitor CI-994 and seven PROTACs capable of degrading HDAC1–3 with different degradation profiles side-by-side .More efforts are needed to investigate the effects of PROTAC-mediated degradation on corepressor complexes. This is a real advantage that the PROTAC-mediated degradation of HDACs could offer over HDAC inhibitors. However, discovering such PROTACs for HDAC1/2 complexes is non-trivial as HDAC1 and HDAC2 exist in six different corepressor complexes interchangeably, with each individual corepressor complex encompassing multiple protein partners. Additionally, the selective degradation of one of the six HDAC1/2 corepressor complexes may have minimal effects on HDAC1 or HDAC2 abundance overall, and such PROTACs could easily be overlooked.63–68 Again, this perhaps reflects the observation in the proteomic study by Xiong et al. that HDAC8 is one of the HDAC isoforms more prone to PROTAC mediated degradation along with HDAC3 and HDAC6. Utilising a previously developed selective HDAC8 inhibitor Chotitumnavee et al. were able to synthesise a selective PROTAC degrader of HDAC8  in comparison to HDAC8 inhibition and degradation by 12 . The authors noted PROTAC 12 also reduced levels of IKZF1 a known neo-substrate for the cereblon E3 ligase, however the E3-ligand pomalidomide alone did not show comparable potency in compromising cell viability. This perhaps suggests that the degradation of HDAC8 by PROTACs may be more effective than the selective inhibition of HDAC8 in compromising cell viability.A number of studies have now been reported on the PROTAC mediated degradation of HDAC8.of HDAC8 .64 PROTAet al. utilised a dual HDAC6-HDAC8 inhibitor for PROTAC design.65 Intriguingly, although the inhibitor from which PROTAC 13 was designed, is a more potent HDAC6 inhibitor than HDAC8 inhibitor, 13 was capable of approx. 30-fold more potent HDAC8 degradation over HDAC6 degradation in HCT-116 cells. 13 exhibited DC50 values of 147 nM and 4.95 μM respectively for HDAC6 and HDAC8. The authors suggest that 13 is more effective at forming a ternary complex with HDAC8 over HDAC6. No degradation of HDAC1 or HDAC3 was observed in the presence of 13. Acetylation of SMC-3 a known substrate for HDAC8 was also observed. Degradation with 13 was time-dependent with degradation initiated after 2 hours, reaching maximum HDAC8 degradation levels at 10 hours, and HDAC8 levels started to rise again at 24 hours. No cell viability studies were reported with 13.Sun et al. synthesised a series of PROTACs designed to selectively target HDAC8 for degradation in neuroblastoma cells, utilising a selective HDAC8 inhibitor as the basis for PROTAC design.66 PROTAC 14 was capable of dose-dependent HDAC8 degradation with a Dmax value (maximum percentage degradation) of 70% for HDAC8 at 10 μM and no degradation of HDAC1 or HDAC6 was observed. Degradation was again also time-dependent, with the maximum degradation of HDAC8 exhibited at 6 hours and recovery of HDAC8 to basal levels noted after 48 hours. Comparative VHL recruiting analogues of 14 exhibited no significant HDAC8 degradation. PROTAC 14 was also capable of increasing the acetylation of SMC-3 and exhibited anti-neuroblastoma activity.Darwish et al. utilised an indole scaffold, again a selective inhibitor of HDAC8, for the development of PROTACs to target HDAC8.67 Unique to this study VHL based PROTACs were capable of effectively degrading HDAC8, however they were again outperformed by cereblon recruiting PROTACs. PROTAC 15 exhibited a DC50 of 0.58 μM for HDAC8 in A549 cells with a Dmax value greater than 95%, and maximum HDAC8 degradation was observed at 18 hours. Again, similar to the results observed by Chotitumnavee et al. PROTACs outperformed the selective HDAC8 inhibitors from which they were originally derived in compromising cell viability in A549 cells. PROTAC 15 was also capable of long duration tumour regression in an A549 tumour mouse model.Huang et al. reported a highly potent HDAC8 targeting PROTAC with a single digit nanomolar DC50 value of 1.8 nM and Dmax value of 97% in MDA-MB-231 breast cancer cell line.6816 was also a degrader of HDAC6 but with a higher DC50 value of 38 nM. Rapid HDAC8 degradation was observed at 4 hours with 16, and HDAC8 abundance was reduced up to 48 hours after treatment. When 16 was investigated in jurkat cells the DC50 value modestly increased to 4.7 nM for HDAC8 and 78.5 nM for HDAC6 highlighting a modest drop in potency and HDAC8 selectivity in a different cell line. Although 16 only exhibited weak anti-proliferative activity in MDA-MB-231 cells it still outperformed the selective HDAC8 inhibitor from which it was derived and also inhibited migration of MDA-MB-231 cells whereby the selective HDAC8 inhibitor did not. In cell viability assays, and assays investigating apoptosis, 16 was more effective than its parent selective HDAC8 inhibitor. However, at the effective micromolar concentrations needed in these assays concurrent HDAC6 degradation with HDAC8 degradation may not necessarily be ruled out.Zhao 69 Li et al. reported that the treatment of HEK293 cells with the proteasome inhibitor MG132 results in an increased abundance of HDAC4, HDAC5, and HDAC7, with HDAC7 levels even further elevated again comparatively to HDAC4 and HDAC5.70 This suggests these HDAC enzymes are prone to proteasome mediated degradation in cells in their own right. Again, this finding correlates well with the proteomic study by Xiong et al.,56 after HDAC6, HDAC3 and HDAC8, the isoforms HDAC7, HDAC5 and HDAC4 respectively were the most frequently identified to be in reduced abundance on PROTAC treatment, with no PROTACs reported to reduce the abundance of HDAC9. In terms of gaining isoform selective degradation by PROTAC treatment within the class IIa enzymes only HDAC4 selective PROTACs have been reported to date.Class IIa HDACs encompass HDAC4, HDAC5, HDAC7 and HDAC9. These HDACs can be found in both the nucleus and cytoplasm and they exhibit significantly reduced deacetylase enzymatic activity on histone proteins compared to class I HDACs.et al. reported the first HDAC4 selective PROTACs to investigate the role of HDAC4 in Huntington's Disease  HDAC inhibitor, reported previously by Scott et al.73Macabuag  Disease .71 They 50 values of the hydroxamic acid-based PROTACs ranged from 28–48 nM with corresponding Dmax values of 77-83%. The TFMO-based PROTACs produced DC50 values ranging from 3–4 nM with associated Dmax values ranging from 72–85%. Interestingly, HDAC4 degradation efficiency was not impacted by varying the PEG linker length. PROTACs 17 and 18, which are representative degraders from each series, were capable of producing a dose-dependent degradation of HDAC4 in Jurkat E6-1 cells. Additionally, PROTAC 17 exhibited the hook effect at 10 μM. By employing multiplexed western blotting, Macabuag et al. evaluated isoform-selectivity of PROTACs 17 and 18 for HDAC4 over other class IIa HDAC isoforms, whereby Jurkat E6-1 cells were treated with the degrader compounds over 24 h at increasing concentrations. PROTACs 17 and 18 exhibited a dose-dependent degradation of HDAC4, while no degradation of HDAC1, HDAC5, HDAC7 or HDAC9 was observed; however, minor degradation of HDAC3 was noted for PROTAC 18 at 10 μM. The study proved that PROTACs 17 and 18 induced HDAC4-selective degradation with no degradation of other class IIa isoforms. Macabuag et al. also demonstrated concentration-dependent degradation of HDAC4 in mouse neuroblastoma cells (Neuro-2a) over 24 h with PROTAC 18.71 However, a 20-fold decrease in degradation efficiency was noted in Neuro-2a cells in comparison to the Jurkat cell line. Yet, again, demonstrating how the cell type can influence the degradation efficiency of the PROTAC.The DCet al. it was discovered that the selective degradation of HDAC6 was observed over the other eleven zinc-dependent HDACs (50 Given this result, and the number of potent HDAC6 PROTACs reported to date in the literature, it does seem that HDAC6 is the most amenable zinc dependent HDAC isoform to proteasome mediated degradation by PROTACs. Of the HDAC isoforms HDAC6 does contain a zinc-finger ubiquitin-binding domain,74 and it is tempting to speculate that this ubiquitin binding domain is a potential reason why HDAC6 is so amenable to proteasome mediated degradation by PROTACs over other HDAC isoforms. In direct comparison there are single digit nanomolar inhibitors reported for HDAC10,75 however to date, as of writing, no PROTACs targeting HDAC10 for degradation have been reported.Class IIb HDACs incorporate HDAC6 and HDAC10. Of the eleven-zinc dependent HDAC enzymes PROTACs targeting HDAC6 for degradation are probably the most frequently reported in the literature to date. When the first hydroxamic acid pan-zinc dependent HDAC inhibitor was incorporated into PROTAC 19 by K. Yang nt HDACs .50 Givenet al. reported the selective degradation of HDAC6 with PROTAC 19 incorporating a pan-HDAC inhibitor as the HDAC ligand,50 An et al. utilised the selective HDAC6 inhibitor Nexturastat A for inspiration in designing PROTACs to target HDAC6.76 PROTAC 20 incorporating Nexturastat A as the HDAC ligand and pomalidomide as the E3-ligand exhibited an impressive DC50 value of 3.8 nM in the B lymphoblast MM.1S. cell line (50 value of 1.21 μM compared to a value of 2.25 μM for Nexturastat A.After K. Yang ell line . No degret al. reported a potent and selective HDAC6 degrader 21 also utilising Nexturastat A as the HDAC ligand,77 with a different linker attachment position to the ligand to PROTAC 20. Again, PROTAC 21 exhibited a single digit nanomolar DC50 value of 1.6 nM in MM.1S cells and could initiate HDAC6 degradation within 1 hour of treatment. PROTAC 21 also degraded IKZF1/3 in a dose dependent manner, known neo-substrates of PROTACs incorporating thalidomide analogues to recruit the cereblon E3 ligase. Above concentrations of 100 nM this dual HDAC6 and IKZF1/3 degradation by 21 was found to be responsible for enhanced antiproliferation effects in MM.1S cells. To abolish IKZF1/3 degradation, K. Yang et al. were able to optimise cereblon recruiting E3-ligands and incorporate them into PROTACs such as 22 to promote the selective degradation of HDAC6 without the co-degradation of IKZFs.78 Highlighting that IKZF degradation does not always have to coincide with PROTACs incorporating cereblon recruiting E3-ligases. In a different study by H. Yang et al., the authors were able to demonstrate that alteration of the linker connectivity to Nexturastat A in PROTAC 23 still maintained potent DC50 values directly comparable to other HDAC6 targeting PROTACs.79Wu et al. reported PROTAC 24 recruiting the VHL E3 ligase to selectively degrade HDAC6 with a DC50 value of 7.1 nM and Dmax value of 95% in MM.1S cells.80 Utilising the VHL ligand for HDAC6 degradation required a longer linker length to target HDAC6 with similar Dmax values to cereblon recruiting HDAC6 PROTACs. One of the potential advantages of 24, similar to 22, is that IKZF1/3 neo-substrates were not degraded when recruiting the VHL E3 ligase and this could be considered a more selective chemical probe for studying HDAC6 related biology. No cell viability assays were reported with 24 in this study.K. Yang et al. designed and synthesised a structurally unique HDAC6 targeting PROTAC 25,81 based on the natural product indirubin as the ligand to engage HDAC6. PEG linkers were explored in this study and intriguingly the shortest linker containing one PEG unit was much more effective at HDAC6 degradation than the longer PEG linkers. 25 exhibited a HDAC6 DC50 value of 108.9 nM in K562 lymphoblast cells with no degradation of HDAC1 observed. The authors were interested in investigating NLRP3 inflammasome activation with 25, and 25 reduced the expression of NLRP3 in THP-1 cells. The selective HDAC6 inhibitor from which PROTAC 25 was derived was less effective in this response, and the inhibitor was also more cytotoxic to cells.Cao et al. utilised their novel solid phase synthesis approach to hydroxamic acid-based PROTACs in an attempt to discover PROTACs to selectively degrade HDAC6.82 They identified 26 and 27 with DC50 values of 3.5 nM and 19.4 nM respectively for HDAC6 in the leukaemia HL-60 cell line. No degradation of HDAC1 or HDAC4 was observed with 26 and 27. 26 and 27 were screened against a small panel of acute myeloid leukaemia cell lines and B-cell acute lymphoblastic leukaemia cell lines for their effects on cell viability. Despite its potent DC50 value for HDAC6, 27 did not compromise cell viability in the cell lines tested at concentrations ranging from 0.5–50 μM, the authors noted that this is an observation similar to others studying the effects of selective HDAC6 inhibitors on cell proliferation.83 PROTAC 26, on the other hand, did compromise cell viability in three of the AML cell lines tested and was capable of inducing apoptosis, but with IC50 values in the double digit micromolar ranges. This was a surprising result given that 26 is not only a selective HDAC6 degrader but also exhibits a similar inhibition profile to class I HDACs as the pan-HDAC inhibitor vorinostat in vitro. The authors hypothesised that the reduced permeability of 26 to the nucleus compared to vorinostat is perhaps responsible for the loss of potency in cell viability assays.Sinatra 84 Offering an alternative to the hydroxamic acid functional group which has previously been reported for mutagenic effects.23 One of the most intriguing findings of this study was that 28 and 29 were compromised in terms of their inhibition of HDAC6 compared to hydroxamic acid HDAC inhibitors, with IC50 values of 0.64 μM and 0.69 μM respectively. Yet, 28 and 29 were more potent degraders of HDAC6 in cells, with DC50 values of 131 nM and 171 nM respectively, both PROTACs exhibited a hook effect at 10 μM. Although extensive studies were not carried out their effects on cell viability, these PROTACs did not compromise cell viability in the MM.1S cells in which they were tested.The same group reported non-hydroxamic acid based PROTACs 28 and 29 that selectively degrade HDAC6 in MM.1S cells.+ to catalyse the hydrolysis of acetylated lysine residues and consist of the sirtuin family SIRT1-7. The first HDAC targeting PROTAC reported in the literature was targeting SIRT2 for degradation.49 PROTAC 30 was synthesised utilising click chemistry and the POI ligand incorporated a selective inhibitor of SIRT2 and thalidomide as the E3-ligand (49 Dose dependent degradation of SIRT2 was observed in HeLa cells with no degradation of SIRT1. SIRT2 degradation by 30 could be further visualised in living HeLa cells with GFP-tagged SIRT2.Class III HDACs utilise NAD3-ligand .49 Dose et al. reported a new SIRT2 inhibitor and incorporated it into PROTACs 31 and 32.8531 and 32 were capable of degrading SIRT2 at concentration range of 0.5–10 μM in MCF7 and BT-549 cell lines, with a hook effect observed at greater concentrations. Via degradation 32 was capable of reducing SIRT2 deacetylase and defatty acylation activity in cells, whereby the SIRT2 inhibitor from which PROTAC 32 was derived was not capable of reducing SIRT2 defatty acylation activity. This could be a major advantage to using PROTACs to degrade SIRTs over inhibition of their catalytic active site alone. At lower concentrations 32 was more effective at compromising cell viability in MCF7 and BT-549 cells than the inhibitor from which it was derived, however at higher concentrations the differences between 32 and the selective SIRT2 inhibitor were much less. The authors hypothesised this is due to the hook effect exhibited on SIRT2 in the presence of 32.J.Y. Hong 86 It is the smallest of the HDAC enzymes and is a much more effective fatty-acid deacylase than a histone deacetylase.87 HDAC11 does not exhibit significant effects on cell proliferation but has been noted as a potential target for metabolic disorders.88 Selective inhibitors of HDAC11 have been reported,89 however as of yet, at the time of writing, no PROTACs targeting HDAC11 for degradation have been reported. HDAC11 could potentially be a very amenable target for proteasome-mediated degradation as Long et al. noted its poly-ubiquitination and short half-life of 4 hours in BEAS-2B cells, which could be reversed in the presence of the proteasome inhibitor MG132.90HDAC11 is the singular HDAC isoenzyme in class IV and was the most recently discovered HDAC in 2002.via the proteasome, rather than solely inhibiting the catalytic active site, will allow researchers to probe the biology of HDACs in cells and in vivo beyond their enzymatic function alone. Five years ago, before the first HDAC targeting PROTAC was reported, this would have been non-trivial, at least by small molecule approaches in chemical biology. It is also clear that the selectivity of degradation is not only PROTAC dependent but also dependent on the cell line, an observation also made by others.91 Before PROTACs are used as chemical probes it would be recommended to first validate the selectivity of HDAC isoform degradation in the cell line of investigation. This seems to be even more imperative when a pan-HDAC inhibitor is incorporated as the HDAC ligand in the PROTAC.It is clear that PROTACs have the capacity to offer potent and selective degradation of individual HDAC isoforms in the cell. PROTACs are already being used as chemical probes to study biology related to individual HDAC isoforms. Degrading the HDAC enzyme et al.56 investigating HDAC isoform degradation in the presence of 48 PROTACs correlates well with the number of isoform-selective HDAC targeting PROTACs reported to date in the literature. PROTACs capable of the selective degradation of HDAC6 are the most frequently reported, followed by selective degraders of HDAC8 and selective degraders of HDAC3. Future challenges will likely involve dialling out the degradation of these isoforms while attempting to degrade another select HDAC isoform. It will also be interesting to witness the discovery of new E3-ligands and how these new ligands can influence or modify HDAC degradation selectivity. In regards to SIRTs much fewer studies have been carried out in comparison to Zn2+ dependent HDACs but the selective degradation of SIRT2 over SIRT1 certainly seems to have been achieved.Some HDAC isoforms certainly seem more prone to PROTAC-mediated degradation than others. The proteomics study carried out by Xiong et al. reported on the noncanonical and non-cytotoxic effects of selective HDAC6 inhibitors in reducing tumour growth in vivo by enhancing antitumor immune responses with immune checkpoint inhibitors in breast cancer.92 Additionally, the class I HDAC inhibitor tucidinostat was found to act synergistically with the aPD-L1 antibody to reduce solid tumours.93 There have been a number of studies recently reported demonstrating how HDAC inhibitors can prime and enhance the anti-tumour immune response of immune check point inhibitors and help overcome associated drug resistance to cancer immunotherapies in solid tumours.94 There are a significant number of active and recruiting clinical trials that are investigating combination therapies of HDAC inhibitors with immune checkpoint inhibitors.94 This could be another novel therapeutic application of PROTACs targeting HDACs. However, the fate of PROTACs targeting HDACs in a clinical context perhaps lies with the fate of exemplary PROTACs targeting AR and ER such as ARV-110 and ARV-471 etc.46 currently in clinical trials and their potential FDA approval status. If and when that time comes, it seems researchers will be primed and ready with a significant number of HDAC targeting PROTACs already available for optimisation to progress to clinical trials.In a therapeutic context, there are examples whereby HDAC targeting PROTACs are more effective at compromising cell viability in cancer cells than their counterpart HDAC inhibitor. There are also examples whereby the reverse is true, particularly when compared to a pan-HDAC inhibitor. However, in future studies, the therapeutic potential of HDAC targeting PROTACs should be investigated beyond cytotoxicity alone. For example, Banik J. T. H. and J. P. S. are inventors on the PCT patent application WO2021148811A1 and the following patents: JP2023-510947, US2023-0120211A1.


As the world population ages, health and social care professionals are increasingly confronted with patients with chronic long-term conditions and multimorbidity, requiring an extensive assessment and integrated care management strategy. The aim of this paper was to systematically collect and assess evidence of interprofessional education and training strategies for Comprehensive Geriatric Assessment (CGA) to build a competent health workforce.A systematic review was conducted according to PRISMA guidelines and the databases Medline, CINAHL, Cochrane and Embase were searched for studies illustrating effectiveness of educational interventions for teaching and training CGA in an interprofessional context.Based on 21 identified studies, a great variability and heterogeneity in duration, setting and design of the interventions was identified. Promising results were found in the domains analysed, ranging from knowledge and skills; practices and behaviour; patient health outcomes; attitudes and perceptions to collaboration and quality of care.Education and training of transversal skills within a continuous learning approach is key to equip the health care workforce for successful CGA performance in an interprofessional environment.Further research in this field is recommended to strengthen the evidence-base towards development of a resilient and integrated health care workforce for an ageing population. Currently, European citizens reach an average life expectancy of 81 years at birth . Actuall3In this regard, the Comprehensive Geriatric Assessment (CGA) constitutes a multi-dimensional diagnostic tool to evaluate the clinical, functional and psychosocial status of geriatric patients and subsequently coordinate and monitor clinical and social care needs and interventions targeting individual resilience . Studies789Due to the multidimensional nature of the CGA, an inter-professional and team-based approach is inherent for its effective and efficient provision in daily practice. The concept of integrated care bundles coherent methods and models to produce collaboration, connectivity and coordination across professional and organizational boundaries . Even ifThe World Health Organization (WHO) advocates for policy strategies strengthening health and social workforce development, with a special focus on collaborative practice 14. In th“students from two or more professions learn about, from and with each other to enable effective collaboration and improved health outcomes” ,has proven to facilitate team-based approaches, preparing students for person-centered care delivery for an ageing population. Despite a growing body of evidence of research in this area, conceptualization, implementation and evaluation of inter-professional educational interventions remain topics in need for further investigation . AgainstBased on the current situation it was the aim of this review to pull together the most recent evidence on inter-professional education and training interventions to teach workforce from different disciplines, thereby facilitating coordinated delivery of CGA in daily practice.This systematic review was conducted in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 checklist 19. The sThe search was limited to publications between January 2000 and September 2022 since contemporary studies rather respond to current evidence-based didactic and pedagogic approaches, thereby also taking into account possible e-learning and digitalization methods.Bibliographic management and deduplication was carried out with the bibliographic software Endnote and manually.In order to be included, studies had to meet the following inclusion criteria: 1) Experimental or quasi-experimental educational intervention studies or cohort studies; 2) applying a multi-professional, undergraduate and/or continuous professional development (CPD) education and training approach in the field of CGA in any setting; 3) and carrying out a self-assessed or objective evaluation of the intervention. Studies were excluded, if the intervention addressed professional silos only and focused on geriatric care in general without referring to geriatric assessments.Outcomes of interest were attitudes and perceptions of learners; knowledge, confidence, competence, abilities or skills; learner’s behaviour and practices; as well as patients’ health outcomes.Each included article was assessed for study quality by two independent reviewers  using the Medical Education Research Study Quality Instrument (MERSQI), a tool developed particularly to assess educational studies consisting of six different domains with an overall possible score range from 0 to 18 . For assNarrative synthesis of the data was performed, as no meta-analysis was carried out due to expected heterogeneity of outcome data.The applied search strategy yielded a total of 379 results, 138 additional results were identified on Google Scholar and via reference tracking. After deduplication (n = 23 results removed), title-/abstract screening (n = 310 results removed) and full-text screening (n = 163 results removed), 21 results were included for synthesis. The PRISMA 2020 flow diagram illustrates the screening process and demonstrates reasons for exclusion .Table 1  summarizes the 21 studies. As illustrated, these studies had been published between 2009 and 2021 with the majority being published between 2013 and 2018. Nearly all of the studies  were conducted in the USA, the rest was undertaken in Greece  and GermIncluded studies represented almost consistently quasi-experimental studies, either with a pre-/post-design [2124252627282930313222343536373840The majority of the studies  considered for this review addressed undergraduate health and social care professions’ students whereas two studies evaluated educational interventions on a CPD level 27. Two p37Disciplines represented in each study were as follows: medicine  [212223242526272829313233343536373821222326272829303134353637392224252627283132343536373824272831323334353637383924252628323439402224262832362426283426322832212728Applied educational and training interventions showed a high degree of heterogeneity, ranging from training courses ; a Geria26233322293032343638Primary outcomes of interest varied as well, with studies analysing overall inter-professional learning experience 243739; a243728362526The majority of the studies used a self-assessment approach of learners [2125262728303132333436373840232924The mean MERSQI score for the included studies was 10.52 (SD 2.07) with a score range from 6.5 to 15.0. Results for each domain indicate a mean score of 1.43 (SD 0.44) for study design, 1.83 (SD 0.51) for sampling, 1.57 (SD 0.85) for type of data, 1.45 (SD 1.13) for validity of evaluation instrument, 2.69 (SD 0.52) for data analysis and 1.50 (SD 0.51) for outcome domain.Primary outcomes investigated in studies included in this review ranged from evaluation of general attitudes and perceptions of the learning experience; knowledge, skills, skill confidence and competencies; practices and behaviour; patient health outcomes as well as other particular measures such as collaborative environment, communication style and quality of care objectives. Attitudes and perceptions in the context of providing geriatric care in an inter-professional environment were analysed in nine studies 2122283036373839.36373821222833363738Positive effects were shown on the development of attitudes towards frailty syndrome and its management in daily clinic towards inter-professional collaboration  and prac3638Mixed results are reported by Renschler et al.  who founEleven studies investigated the domains knowledge, skills, (skills) confidence and competencies [21252627293031343540212527293134352630Turrentine et al.  describe2527Mixed results are reported in terms of (skills) confidence , self-asEveryday practices and team behaviour were analysed by two studies included in this review 24. The ePatient health outcomes were analysed by one study and are briefly presented here . ConsideIn terms of collaboration and quality of care, a randomized controlled trial conducted with medical and nursing students reported a post-intervention increase of evaluated care objective scores in the intervention and control group in all but one category: care of other symptoms. The intervention group achieved significant post-intervention scores in three categories: pain therapy (p = 0.006); guarding of patient’s autonomy (p = 0.001) and integration of psychological aspects (p = 0.003). Moreover, a significant change in interaction initiation was observed in the intervention group (p = 0.0007) and there was a significant post-intervention increase in the number of uni-professional information items exchanged in both groups (p < 0.0001) and in subgroups (p = 0.0002-p = 0.004) .Byerly et al.  additionDefinition and development of transversal skills of tomorrow’s health workforce will be key to enable different professions in health and social care to deliver high quality and patient-centred integrated care in health care systems of the future 44. SkillBased on the concept of ICP and “adaptive expertise”, the current paper describes the evidence-base for trainings offered for different health professional groups to perform CGA in inter-professional collaborative practice.The overall care rendered by CGA teams providing longitudinal assessment and care can be divided into six steps: data-gathering, discussion among the team, increasingly including the patient and/or caregiver as a member of the team, development of a treatment plan – together with the patient and/or caregiver, implementation of the treatment plan, monitoring response to the treatment plan and finally revising the treatment plan . Given t32The need for targeted inter-professional education and training initiatives in geriatric care has been highlighted by literature for some time 5152. Ind5153Studies included were of varying quality in study design, only one study using a randomized controlled trial approach design . DuratioGiven the heterogeneity of IPE interventions published in the papers included and this major weakness in the evaluation frameworks applied to evaluate the educational impact of training interventions, the data provided in this review should be reflected thoroughly. Two of the studies included 39 set upResults of this review need to be considered in light of its limitations. As already mentioned, the studies included present with a strong heterogeneity in terms of study design, outcomes and evaluation methodologies. This hampers the possibility to present a clear evidence-base and only allows drawing a cautiously positive picture of effectiveness of educational interventions to train CGA across health and social care professions, which is aggravated by the fact that not all studies provided full disclosure of educational content delivered and intervention execution. Most of the studies included presented self-reported data based on learners’ perceptions instead of objective assessment results or patient health outcomes that may lead to questioning reliability and generalizability of effects.Despite the mentioned heterogeneity, the majority of studies included encompasses didactic approaches with a strong practical orientation, either in form of real patient encounters [222425303233363738262829313923The data on training formats, however, contrast with assessment methods applied as only two studies assessed practices and behaviour 24 and onNone of the studies used the Objective Structured Clinical Examination (OSCE) as assessment methodology. The OSCE is a widespread method and gold standard to assess clinical skills and competencies transmitted during educational and training initiatives as objectively as possible 57. Despi131560Although authors of this review adhered to guidelines for writing systematic reviews 19, some This systematic review highlights effects and possibilities to educate and train professionals in health and social care in conducting a CGA and shows promising results for inter-professional collaborative practice in geriatric care, when measured with students’ self-assessment scales. There seems to be potential for introducing case-based and/or work-placed teaching methodologies reflecting the complexity of interacting physical, psychological/emotional, contextual and social factors evaluated during CGA for different professions, enabling a shared decision process during CGA of older people.However, further educational research, preferably reflecting effects, needs and progress of interprofessional collaborative practice in the European education/training landscape and focussing on effectiveness of complex care based training situations based upon standardized educational assessment frameworks is needed to strengthen the evidence-base, foster education and training standards as well as curricular development in an inter-professional context and thus adequately equip the health workforce to acquire adaptive expertise for delivery of CGA and, therefore, meet the needs of an ageing population.The additional file for this article can be found as follows:10.5334/ijic.7549.s1Supplemental material.Research protocol.


In hereditary spastic paraplegias (HSPs), magnetic resonance imaging (MRI) scans of the brain typically show the involvement of the anterior part of the corpus callosum with abnormalities in the white matter fibers of the forceps minor(“ears of the lynx sign”). However, these imaging findings are particularly associated with spastic paraplegia type 11 (SPG11) or spastic paraplegia type 15 (SPG15).NT5C2(p.[Leu468Pro] and p.[Gln542Argfs*71]), consistent with SPG45, of the SPG subtypes with thin corpus callosum.A child with spastic gait achieved independent walking at the age of 2 years, and 3 years later was referred to our service for investigation. A brain MRI demonstrated corpus callosum dysgenesis and peritrigonal white matter abnormality . Whole


Following the publication of this article , the corThe 2D gel electrophoresis image in Figure 1A in , and FigThe shVector panels in Figure 6A of  are simiThe shVector panel in Figure 7A of  is similIn the Actin panels in In ○ The xenograft tumor study is described as having 3 male mice per group, but the corresponding authors clarified that 2 male and 2 female mice were used per group.○ 8% and 10% SDS-PAGE gels were used for western blot experiments, not 12% as stated.○ Sequences of shRNAs against ALDOA and the non-targeting control are absent from the article. The corresponding authors provided the relevant sequences in The following inaccuracies/omissions were identified in the Methods section:The corresponding authors stated that data underlying all the results reported in the article are available, except for the raw image data underlying the western blots in Figure 5.The corresponding authors confirmed that the same 2D gel image and cohort of 7 patients are reported in four articles –4, and eThe underlying image data for the actin panels in For PLOS ONE Editors issue this Expression of Concern to notify readers of the above issues and relay the supporting data and updated figures provided by the corresponding authors.The S1 File(DOCX)Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(ZIP)Click here for additional data file.


Poly(pyrazolyl)borate ligands have been obtained throughthe reactionof highly reactive haloboranes with in situ formed pyrazolides undervery mild conditions. This versatile synthetic method allows the selectivesynthesis of bis-, tris-, or tetrakis(pyrazolyl)borates. Furthermore,the method is compatible with the use of functional groups on theheterocyclic moieties of the poly(pyrazolyl)borates that were notaccessible to date. Strongly encumbered sodium and thallium(I) poly(pyrazolyl)borateswith a reduced donating ability have been obtained for the first time. Since then, poly(pyrazolyl)boratesof almost any transition metal have been prepared4 and used as enzymatic models,5 for the development of new materials,6 and as power catalysts in reactions as carbene or nitrene C–Hinsertion, polymerization or carbonyl derivatizations.7 The success of this family of ligands resides in the possibilityof fine-tuning the electronic and steric properties of the metal complexesthrough the introduction of appropriate groups on the heterocyclicrings. However, despite the more than 4200 crystal structures of thistype of complexes that have been described,8 the simultaneous pyrazole decoration with bulky and electron-withdrawingsubstituents has not been possible to date.Poly(pyrazolyl)borate ligands were describedfor the first timeby Trofimenko during the 1960s.9 This transformationpresents drawbacks: (i) difficult control of the reaction stoichiometrywith possible formation of mixtures of dihydrobis(pyrazolyl)borates(Bpx), hydrotris(pyrazolyl)borates (Tpx) andtetrakis(pyrazolyl)borates (Tkpx), (ii) hazardous evolutionof hydrogen gas under high temperature conditions,10 (iii) limited functional group scope due to their sensibilityunder reductant conditions and (iv) pyrazoles containing simultaneouslyelectron-withdrawing and bulky substituents do not react under theseconditions due to reduced nucleophilicity and higher steric hindrance.The access to Tpx ligands presenting these characteristicscould widen the catalytic applications of their metal complexes dueto the increased electrophilicity,11 easierreduction,12 and higher stability of lowoxidation states13 of the correspondingmetal centers.The most commonroute to prepare poly(pyrazolyl)borates is thereaction of a high excess of the desired pyrazole derivative witha metal borohydride in the absence of solvent 1a.9 This14 very flammable reagents,in a reaction with the same disadvantages related before boratesBCl2 with nonsubstituted sodium pyrazolide.17The third generation of tris(pyrazolyl)boratesappeared with thesubstitution of the hydrogen atom with an alkyl or an aryl moiety.Alkyltris(pyrazolyl)borates were prepared from lithium alkylborohydridederivatives,d before 1b. In th)borates1c.16 AltHere we present a new practical and directmethodology for thepreparation of poly(pyrazolyl)borate ligands under very mild conditions,with wide applicability that yields exclusively the desired poly(pyrazolyl)boratederivative in good to excellent yields 2.3Tl), the usual entry for metalexchange.4a A dichloroborane dimethylsulfidecomplex (BHCl2·SMe2) was chosen as thehighly reactive boron source and 3-tert-butylpyrazole(1a) as the standard azaheterocycle borate(3aNa) just using the ca. stoichiometric amounts of all reagents on toluene atroom temperature for 24 h. Conditions optimization for tris(pyrazolyl)borateligands synthesis has been performed and is specified in the 3aTl was prepared in situ through standard proceduresto facilitate product purification.14aFirst, we focused on more extended thalium(I) tris(pyrazolyl)borates(erocycle 3. A baseerocycle . A smootxTl (3a–jTl) using the optimized reaction conditions. An excellent 90% yieldwas obtained for complex 3bTl bearing the encumbering tert-butyl group at position3 and a bromine atom at position 4 of the heterocycle. This methodallows for the first time the preparation of boron scorpionate ligandswith a strongly electron-withdrawing nitro group in the presence ofa bulky substituent such as tert-butyl. Ligand 3cTl was prepared in a satisfactory93% yield. Introduction of the bulkier adamantyl group at position3 (3dTl) did not negatively affectthe product yield. All attempts to prepare 3bTl, 3cTl and 3dTl presenting electron-withdrawinggroups and highly hindering tert-butyl or adamantylsubstituents through literature methods were unsuccessful.9b The preparation of TpxTl bearing a mesityl group on position3 also proceeded smoothly in good yields with a hydrogen (3eTl) or a bromine (3fTl) atom on position 4 of the pyrazole ring.These optimized conditions were used in thepreparation of thalliumhydrotris(pyrazolyl)borate complexes using the selection of pyrazolederivatives, as shown in 1g bearing asensitive aldehyde substituent at position R4. As expected,the reaction proceeded smoothly, and the corresponding sodium complex 3gNa was obtained in 79% yield. Finally,we challenged the scope of the method using as the starting materialthe trisubstituted pyrazole 1h containing two methylgroups and one electron-withdrawing nitro group. The expected product 3hNa was obtained in a satisfactory86% yield. For pyrazoles 1g and 1h sodiumtris(pyrazolyl)borates were obtained directly, and sodium to thalliumexchange was not performed. The introduction of two encumbering isopropylgroups hinders the formation of 3iTl, and only traces of the expected product could be detectedeven at higher temperatures.We have also explored the compatibility of this newprocedure forthe synthesis of pyrazolylborates supporting functional groups ofspecial sensibility to reductant environments. We used our standardconditions in the reaction with pyrazole 1a–g ,18 difficult to isolate as pure materials.19 The results obtained using acommercially available chloroborane dimethylsulfide complex (BH2Cl·SMe2) for a selection of thallium(I) Bpx complexes are shown in Tl3 were used, and pyrazole and base equivalents were adjustedto ca. ideal stoichiometric amounts with good results. Pyrazoles 1a–c bearing a highly encumbering tert-butyl group cleanly produced the expected 2a–cTl in good to excellentisolated yields. In these cases, the isolation of compounds 2a–cTl is greatlyfacilitated by our selection of the boron source that allows fullcontrol of the 2:1 stoichiometry, thus avoiding contamination withother pyrazolylborates. However, the use of the standard conditionsfor 2e–f yielded a complex reactionmixture due to the formation of the desired 2e–fTl besides the two correspondingpyrazaboles with two bridgehead boron atoms borates borate.The scarce examples described of these ligands are almost restrictedto those presenting methyl or hydrogen groups on positions 3 and/or5 of the pyrazole rings due to the difficulty associated with thethermal introduction of the fourth heterocycle.22xNa (4a–fNa) with highlyencumbering substituents. Alkaline salts of these ligands are a commonentrance to other metal complexes through metal exchange.22Further, we extended our procedure for the preparationof sterically challenging and rare tetrakis(pyrazolyl)borates.2cTl, 3bNa(OH2), and 4fTl were determined by single crystalX-ray diffraction borate ligands with excellent yields andcomplete regioselectivity.In conclusion, we have developed a usefuland versatile new syntheticprocedure for the selective preparation of Bp


Ab initio molecular dynamics (AIMD) simulations show the significance of asymmetric motion on a multidimensional potential energy landscape around the metal center for activated crossover from triplet ligand centered (3LC) to triplet metal centered (3MC) states on picosecond timescales. Significant entropic differences arising from the structural distributions of the 3LC and 3MC states revealed by the simulations are found to favor the forward crossover process. Simulations at different temperatures provide further insight into the interplay between structural and electronic factors governing the 3LC → 3MC dynamics as a concerted two-electron energy transfer process, and the broader implications for photoinduced generation of high-energy 3MC states of interest for emerging photocatalytic applications are outlined.Excited state evolution of the rhodium( iii) complex [Rh(iii)(phen)2(NH3)2]2+  has been investigated theoretically to gain a better understanding of light-driven activation of high-energy metal centered states.Excited state evolution of the rhodium( One strategy to overcome this limitation is to activate high-energy 3MC states indirectly through energy transfer from another chromophoric unit with better light-harvesting capabilities.8 This can be achieved as an intramolecular activation from an initially excited CT or ligand centered (LC) state. Another challenge with the involvement of MC states in d6 complexes is the population of anti-bonding eg orbitals that frequently causes substantial excited state energy losses, associated with weakened metal–ligand (M–L) bonds.15,16 Activated crossover into high-energy 3MC states following initial LC excitation is, however, known from studies of prototype Rh(iii) complexes, as evidenced by experimental observations of dual photoluminescence (PL) from both the 3LC and 3MC states, as well as photochemical quenching experiments involving the 3MC state.17–26 The effect on the relative energy between the 3LC and 3MC and its impact on the state-to-state crossover efficiency was studied experimentally for a series of tris-phenanthroline derivatives, with the general formula [Rh(iii)(phen)2XY]n+ , by tuning the ligand field strength.19Transition metal complexes that combine efficient light-harvesting with long-lived, high-energy excited states are widely used in molecular photocatalysis and related photochemical applications.27–35 Some simulations have also covered the explicit interplay between excited state deactivation processes and solvent relaxation and reorganization processes.36Quantum chemical simulations offer significant opportunities to gain detailed insight into several different aspects of the excited state evolution of transition metal complexes. A variety of many-state quantum dynamical simulation methods have provided important insights into the ultrafast dynamics associated with initial relaxation processes within a dense manifold of excited states.3LC → 3MC state crossover activation in the prototype Rh(iii) phenanthroline complex, [Rh(phen)2(NH3)2]3+, using ab initio molecular dynamics (AIMD) simulations conducted at the density functional theory (DFT) level. The [Rh(phen)2(NH3)2]3+ complex provides a good prototype system to investigate such 3LC → 3MC crossover dynamics computationally as experimental observations of dual emission in the 170–200 K temperature range point to a favorable 3LC–3MC balance, similar to what has also been reported for the [Rh(iii)(phen)3]3+ complex.19,24,37,38 We focus on intramolecular aspects of activated 3LC → 3MC crossover dynamics on picosecond timescales, i.e. excited state dynamics taking place on the lowest excited triplet potential energy surface following any initial ultrafast ISC and IC processes as well as initial local relaxation of the initially populated 3LC state. This provides a largely complementary set of computational challenges compared to many recent studies focused on the earliest ultrafast multi-state relaxation processes, and places a greater focus on accounting for large-scale structural distortions on a complex multidimensional molecular energy landscape on timescales that can extend to tens of picoseconds and longer.In this work, we investigate the 23LC and 3MC states in the [Rh(phen)2(NH3)2]3+ complex were performed at the unrestricted DFT (uDFT) level of theory using the B3LYP* hybrid functional with reduced 15% Hartree–Fock exchange (HFex) and the 6-311G basis set with the Stuttgart–Dresden SDD effective core potential for the central Rh atom.39–42 An implicit Polarizable Continuum Model (PCM) was applied to account for a water solvent environment.43 The nature of the various states was confirmed by frequency analysis. All calculations were conducted using the Gaussian09 software.44Full geometry optimizations of structurally distinct relaxed ex)/6-311G/(water) in the 3MC and 3LC excited states were chosen as starting geometries for subsequent AIMD calculations using the atom centered density matrix propagation (ADMP) model.45 The size of the basis set was reduced to 3-21G in order to reduce the computational costs, and motivated by test calculations indicating only minor differences for the calculated energy differences between the relaxed 3LC and 3MC states according to a comparison with the larger 6-311G basis set. Explicit solvent structure and dynamics were neglected in this study focusing mainly on intramolecular rearrangement aspects, and an overall solvent response was approximated with the PCM model. The temperature dependence of the dynamics was investigated at constant selected temperatures  imposed by a standard thermostat. A minimum energy path was calculated as a complementary way to track the 3MLCT → 3MC conversion (details in ESIOptimized structures at the B3LYP*(15% HF33.1iii)(phen)2(NH3)2]3+ complex from a first exploration of the triplet PES by means of unconstrained geometry optimizations and single point quantum chemical calculations. A single 3LC state minimum, identified as a local excitation on one of the phenanthroline ligands (designated as phen′), as well as a set of four non-degenerate local minima of 3MC nature were identified by geometry optimizations. Different orientations of the eg orbitals relative to the M–L bonds allow for up to six different 3MC states associated with Jahn–Teller distortions along different M–L axes. The C2 rotation axis, characteristic of the C2 point group to which [Rh(iii)(phen)2(NH3)2]3+ complex belongs more specifically, reduces the number of unique 3MC states to four. A graphical representation of the eg orbital orientations along different M–L bonds, together with the names for the corresponding states adopted in this report , are described in g orbital set more strongly compared to the homoleptic parent complex [Rh(iii)(phen)3]3+. This potentially increases the energy offset between the distinct 3MC states. Indeed, different emission band broadening characteristics for the two Rh complexes of ∼40 cm−1 in acetonitrile at 297 K for [Rh(iii)(phen)3]3+ and ∼60 cm−1 in a water/ethylene mixture at 170 K for [Rh(iii)(phen)2(NH3)2]3+ provides a likely experimental indication of such differences in the MC state PES landscapes.19,38Several local triplet state minima were identified for the 3+ complex, with the aim to reveal the crossover dynamics as well as further properties associated with the population dynamics of thermally accessible states.The M–L bonds of the relaxed 3.2iii)(phen)2(NH3)2]3+ after initial optical excitation and intersystem crossing into a 3LC excited state, we initially performed AIMD calculations at 170 K, 200 K, and 298 K.To better understand the triplet state dynamics in 2+ complex has, for example, also been reported at the uDFT level.48 All the bond elongations and contractions at 200 K suggest the population of a single dx2−y2 d-type-orbital, and the M–L bond values also correlate well with the calculated distances in the fully relaxed 3MC1 excited state.M–L bond distance distribution plots are analysed in 3MC excited state is populated in this simulation. Two bond distributions with average distances of ∼2.1 Å (blue and orange peaks) are shorter than the remaining four coordination sites in agreement with an eg-dx2−y2 orbital assignment characteristic of the 3MC2 state  to 10.26 D (red peak) corresponding to the 3MC2 state (170 K simulation). Both 3MC1 and 3MC2 calculated dipole moments by uDFT minimization calculations (8.08 D and 10.74 D) reinforce the general state assignments, and the electronic identity of each 3MC state. Larger peak broadening additionally suggest that the 3MC1 is able to move over a larger accessible volume in the multidimensional PES compared to the 3MC2 state.A clearly different set of M–L bond distance distribution profiles top  at the tC2 state . Other pFig. SI.7. The geo3MC dynamics at 298 K. At this higher temperature, the M–L bond distributions access a wide range of distances during the simulation, and with more than one distribution peak. Furthermore, the dipole moment histogram at 298 K shows a bimodal distribution characteristic of alternating population of two relatively distinct local electronic states  are registered, mainly in the Rh – N1 bond reaching a distance of 2.23 Å right before the transition occurs. αSOMO orbital , after temporarily reaching a maximum Rh – N bond stretching of 2.92 Å. This particular initial distortion could potentially represent a sweet spot for initiating photochemical reactions.First, an AIMD simulation at 330 K shown in the top panel of al 3a in  at the d3LC lifetime was extended significantly to ∼17 ps in order to successfully capture a crossover event.Triplet crossover dynamics was also simulated at a lower temperature of 298 K as shown in 1 bond stretching to 2.6 Å. Although the αSOMO orbital 2b shows population of the 3MC3 state rather than the 3MC4 observed in the previous simulated temperature, the 3LC excited state is still deactivated through a higher energy MC state. This initial population of the 3MC3 state is rapidly converted into the 3MC4 excited state in ∼900 fs . An observed second populated 3MC4 state achieves even longer lifetimes up to ∼2300 fs (tIb mark), before finally deactivating to the calculated lower energy 3MC2 excited state (tIIb mark).While the limited simulation data precludes a reliable Arrhenius analysis that can be correlated quantitatively with the experimentally observed temperature-dependence, it is already notable that the simulations over a range of moderate temperatures shows a clear temperature-dependence of the crossover rate. Furthermore, the transition times in the 1–20 ps time range are consistent with the crossover being an activated process with a small effective transition barrier in basic agreement with a relaxed minimum energy path . The metal center activation can in principle be achieved in a concerted or step-wise fashion via electron hopping followed by hole hopping or the equivalent reverse path as illustrated in 3LC → 3MC mechanism in detail from the simulations, the weights of the molecular orbital coefficients from the metal d-atomic-orbitals in the αSOMO and beta semi-unoccupied molecular orbital (βSUMO) were tracked for a series of snapshots with an interval of 4 fs during a 400 fs time window around the transition region  than at 298 K so that the transition state (TS) is essentially reached without any initial geometry stretching. In contrast, a TS at 298 K is only reached by bond stretching after significantly longer state sampling. The two highest energy 3MC excited states behave as important intermediate states, reflecting the complexity of multi-step deactivation paths towards the ultimate population of the lowest 3MC2 state.Finally, the AIMD simulations also highlight the importance of statistical factors influencing the crossover dynamics beyond what can be extracted from static calculations of transition barriers and scans of reaction paths. In particular, the simulations indicate a clear significance of entropy factors related to the accessible PES volume for the different states. This effective area, as an intrinsic characteristic of the distinct excited states dynamics for both sampled temperatures, are already revealed in the trajectories within the phen M–L bonds during the simulation time as represented in 3MC product state corresponds an increase of the reaction entropy. Since the 3LC → 3MC transition is mainly ruled by M–L Jahn–Teller distortions, we assess the change in accessible coordinate space in the simulations by the six M–L bond distributions. In this approximation, the accessible volume of each state can be expressed as the product of the standard deviations of each M–L bond (δli) byThe larger accessible volume in the Sr) then relates with the Boltzmann constant kB and the partition function in terms of volume changes between the 3MC state (VMC) and the 3LC state (VLC) asThe entropy change of the crossover reaction (Δiii)(phen)2(NH3)2]3+ complex together with the final active volume V (phen)2(NH3)2]3+ on picosecond timescales. Initial results from quantum chemical calculations revealed a clear difference in the triplet potential energy landscape between well-defined 3LC states on the phen ligands on one hand, and a more complex 3MC potential energy landscape comprising several local minima with distinct structural and electronic characteristics on the other hand.AIMD simulations were used to investigate 3LC internal state dynamics reveal a narrow and well-defined range of accessible geometries closely constrained near the ground state geometry. In contrast, 3MC dynamics is revealed to involve significant structural mobility on a flat 3MC PES that includes facile transitions between several Jahn–Teller distorted minima. The MC dynamics notably includes fast transitions through higher-energy MC states connecting different low-energy parts of the full 3MC PES. Notably, the large difference in thermally accessible coordinate space between the LC and MC parts of the triplet PES indicates that entropic effects will have a significant influence on the free energy balance and effective driving force governing the 3LC → 3MC crossover dynamics.The AIMD simulations provide further insight into the multidimensional dynamics taking place on the complex triplet PES. First, 3LC → 3MC process at different temperatures showed a significant influence of the temperature on the crossover rate, consistent with an activated process characterized by a small energy barrier. The simulations also reveal further details of the triplet dynamics proceeding via a complex cascade involving several local 3MC states following the initial crossover from the 3LC state. Analysis of the AIMD simulations also enabled an in-depth assessment of the electric rearrangements taking place during the 3LC → 3MC crossover. In particular, the 3LC → 3MC transition is seen to involve a rapid redistribution of both the excited electron and the accompanying hole from one of the phen ligands to the metal center that takes place in a fully concerted fashion on a sub-picosecond timescale. This corresponds to a spin-allowed intramolecular two-electron excitation energy transfer process that takes place dynamically as a structure-driven weakening of the ligand field splitting of the metal center. It is interesting to consider as a topic for further investigation that the two-electron nature of this process appears to provide a fundamentally different set of opportunities and limitations for tuning of the excited state landscape compared to the corresponding one-electron case encountered in more widely studied 3MLCT → 3MC deactivation processes.Simulations of the 3LC → 3MC transition provides useful insights for further developments of transition metal complexes that aim to utilize 3MC states for photochemical applications more broadly. In particular, 3LC → 3MC crossover is confirmed to provide a promising approach to overcome problems associated with weak absorption and ultrafast initial energy losses from direct excitations into 3MC states more generally, as long as the full complexity of the triplet potential energy landscape and associated dynamics revealed by simulations is taken into consideration.Finally, the in-depth understanding of this αSOMO 3LC → 3MC transition dynamics MP4 movie at 298 K is also available in the ESI.†DFT energies and geometry details, trajectories and electron structure analysis of the MD simulations, NEB calculation analysis, and cartesian coordinates of the optimized structures are available in the ESI.PP supervised the project and IBL performed the calculations. PP and IBL contributed to the manuscript preparation.There are no conflicts to declare.SC-014-D3SC04381A-s001SC-014-D3SC04381A-s002


Flt4 knockout mice to investigate VEGFR3 signaling in lymphatic junctions. We show that loss of Flt4 prevents specialized button junction formation in multiple tissues and impairs interstitial absorption. Knockdown of FLT4 in human lymphatic endothelial cells results in impaired NOTCH1 expression and activation, and overexpression of the NOTCH1 intracellular domain in Flt4 knockout vessels rescues the formation of button junctions and absorption of interstitial molecules. Together, our data reveal a requirement for VEGFR3 and NOTCH1 signaling in the development of button junctions during postnatal development and may hold clinical relevance to lymphatic diseases with impaired VEGFR3 signaling.Lymphatic capillaries develop discontinuous cell-cell junctions that permit the absorption of large macromolecules, chylomicrons, and fluid from the interstitium. While excessive vascular endothelial growth factor 2 (VEGFR2) signaling can remodel and seal these junctions, whether and how VEGFR3 can alter lymphatic junctions remains incompletely understood. Here, we use lymphatic-specific   Lymphatic capillaries form discontinuous junctions called buttons to enable absorption of interstitial contents. Jannaway et al. show that vascular endothelial growth factor receptor 3 (VEGFR3) expression is required for the formation of button junctions and that NOTCH1 signaling can fully rescue button formation in the absence of VEGFR3. However, inherited genetic mutations can impair lymphatic vessel function and lymph flow to cause a collection of diseases known as congenital lymphedema. While several gene mutations have been identified in the past decade in congenital lymphedema patients,3 Button junctions first begin to form at E17.5 in the skin lymphatic vasculature and mature by 3 weeks after birth.4 While angiopoietin-2 and DLL4 signaling in lymphatic endothelial cells (LECs) regulate button junctions in lymphatic capillaries in the skin and intestine, respectively,6 the molecular mechanisms required for button formation during development are still poorly understood. Recently, genetic deletion of two VEGFA receptors, Flt1 and Nrp1, from vascular endothelium resulted in the zippering of existing intestinal lacteal buttons and prevented high-fat-diet-induced obesity in mice.7 The proposed cause of this zippering was increased VEGFA ligand availability and thus excessive activation of VEGFR2. This role for VEGFR2 was confirmed by a new model of viral infection that caused button junctions to revert to continuous zipper-like junctions.8 In contrast, a clear mechanism for VEGFR3 in button formation has not been elucidated.To prevent or clear edema, lymphatic vessels absorb the majority of interstitial fluid. Interstitial fluid absorption by lymphatic capillaries is permitted via the remodeling of continuous junction proteins, reminiscent of zippers, into punctate discontinuous junctions that have been referred to as buttons.Flt4 from all LECs postnatally (Flt4iLECKO). Our data reveal that VEGFR3 is required for button formation during a critical period of postnatal development but is later dispensable for button junction maintenance. We also show that overexpression of the NOTCH1 intracellular domain (NICD) is sufficient to rescue button formation in the absence of VEGFR3 and restores the function of lymphatic capillaries to absorb interstitial fluid. Our findings reveal that a VEGFR3/NOTCH1 signaling axis is required for button formation and interstitial absorption. Furthermore, the most common form of congenital lymphedema, Milroy disease, is caused by dominant-negative point mutations in the VEGFR3 gene, and these patients exhibit dysfunctional interstitial absorption. Thus, our findings here hold potential clinical relevance to this disease.Here, we identify VEGFR3 as a major regulator of button junction development during postnatal life. To better understand the role of VEGFR3 in lymphatic function, we induced the deletion of Flt4 was deleted from LECs by crossing Flt4fl/fl9 with a lymphatic-specific Prox1CreERT2 strain.10 While this Cre strain can delete genes highly efficiently with two injections of tamoxifen (TM),12 we and others have found that inactivation of Flt4 led to the reappearance of VEGFR3-competent LECs.13 Therefore, we administered TM every other day from P1 until analysis at P21 . To assess button junction development, we located tissues rich in lymphatic capillaries that remodeled their junctions into buttons. The lymphatic vessels of the diaphragm were previously used as a model for postnatal button formation.5 All lymphatic vessels in the diaphragm of control and Flt4iLECKO pups expressed the lymphatic capillary marker LYVE1  and fewer buttons (<1% vs. 60%) compared to littermate controls, indicating that VEGFR3 is required for button formation in the ear skin  expression in both the blood17 and lymphatic13 vasculature. In blood and lymphatic endothelium, DLL4 is the ligand for NOTCH receptor 1 and 4.19 We found that the expression of DLL4, NOTCH1, and NOTCH4  is cleaved and translocates to the nucleus to initiate gene transcription.20 Western blot for cleaved NICD1 and NICD4 in shFLT4-treated cells revealed that NICD1 activation was significantly decreased, whereas NICD4 had very low basal expression that did not change . Kdr was deleted from P1 with the same dose and frequency of TM as before , simultaneously deleting both receptors from P1   , signifi to ~3%) ; 36%–17% to ~3%) ; 11%–79%nockouts  and S5I Flt4iLECKO mice. Thus, we bred Flt4iLECKO mice with Rosa26-NICD1 mice  that express the NOTCH1 NICD, which induces constitutive NOTCH1 signaling after Cre recombination21  in the Flt4iLECKO mice. The NICD1 fragment also rescued the ability of lymphatic capillaries to absorb interstitial tracer injected into the ear. Finally, our data show that NOTCH1 signaling can accelerate button formation, so NOTCH1 should be investigated in future studies as a potential inducer of button formation.This study demonstrates that VEGFR3 is required for button junction formation and interstitial fluid absorption in multiple tissues. Because genetic deletion of VEGFR3 at a developmental time point when buttons are fully formed does not affect the number or morphology of buttons, we conclude that VEGFR3 signaling does not maintain button junctions and, further, that there is a critical postnatal developmental window wherein VEGFR3 must be activated to successfully form buttons. To investigate the mechanisms downstream of VEGFR3 activation, we cultured hdLECs with a 22 although these findings were challenged by studies suggesting that VEGFR2 only weakly activates DLL4 and instead proposed a role for VEGFR3 activating DLL4 and NOTCH signaling.24 In lymphatics, loss of VEGFR3 was shown to decrease DLL4 expression, which in turn decreased NOTCH1 signaling.13 Surprisingly, we found that DLL4 levels did not change at the protein level, although NOTCH1 activation was significantly decreased in FLT4 knockdown hdLECs. Importantly, we show that forced activation of NOTCH1 signaling in the absence of VEGFR3 restores the ability of lymphatic capillaries to form buttons and absorb interstitial fluid, identifying NOTCH1 as a potential druggable target even in the absence of VEGFR3. Future studies will be needed to probe which NOTCH ligands are inducing NOTCH1 cleavage and whether NOTCH ligands are tissue specific in the context of button formation.While previous studies have demonstrated that VEGFR3 and NOTCH receptor signaling are closely linked, a direct role for these specific receptors in button formation has not previously been demonstrated. VEGF-A/VEGFR2 signaling was shown to induce DLL4-NOTCH signaling during angiogenic sprouting,FLT4 gene cause the most common type of congenital lymphedema, called Milroy disease.26 Milroy patients completely fail to absorb interstitial tracer during a lymphoscintigraphy exam, similar to our Flt4iLECKO mice here.27 Given the known role of VEGFR3 as the main receptor required for lymphangiogenesis,28 it was first suspected that dermal lymphatic vessel aplasia caused Milroy disease.29 However, this idea was challenged when skin biopsies from the feet of Milroy patients were found to contain lymphatic vessels.27 Most mutations in FLT4 occur in the kinase domain, thereby abolishing the kinase activity of VEGFR3.30 Because VEGFR3 makes homodimers or heterodimers with VEGFR2 to signal, these point mutations act in a dominant-negative manner.32 The lack of a phenotype in the Kdr knockouts here argues against heterodimers being involved in button formation. Mice with a similar point mutation in Flt4, known as Chy mice, are a mouse model of congenital lymphedema.29 Thus, the Flt4iLECKO mice reported here that lack VEGFR3 protein likely mimic the dominant-negative mutations in the Milroy patients or Chy mice that have very low residual VEGFR3 signaling. Based on our present data, we hypothesize that a failure to form buttons may underlie the pathogenesis of the Milroy patients and that future work is needed to investigate our signaling mechanism in the Chy mice.Heterozygous point mutations in the 5 and mutations in ANGPT2 have since been identified in congenital lymphedema patients.33 Interestingly, a recent study found that inhibition of angiopoietin 2 or deletion of the angiopoietin receptors, Tie1 and Tie2, decreased the expression of VEGFR3 in LECs.34 However, to our knowledge, lymphoscintigraphy was not performed on these patients, so it is unknown whether they fail to absorb interstitial tracer and thus if defective button formation contributes to this form of lymphedema. Together with the findings here, it argues that a single signaling pathway, namely ANGPT2/VEGFR3/NOTCH1, is responsible for regulating button junction formation.Angiopoietin 2 is also required for button formation,35 A recent study found that acquired lymphedema patients have elevated levels of the proinflammatory mediator leukotriene B4 (LTB4),36 which inhibited VEGFR3 expression and phosphorylation and inhibited NOTCH signaling in LECs. Thus, it is tantalizing to speculate that the lymphatic capillaries of acquired lymphedema patients may have aberrantly zippered junctions as part of the disease progression. Further supporting our data, Tian et al. also showed that lymphatic-specific deletion of Notch1 prevented the absorption of an interstitial tracer and prevented a pharmacological rescue of tail lymphedema, indicating that NOTCH1 signaling must remain intact for LTB4 inhibition to be effective at preventing lymphedema. Given that we show here that overactivation of NOTCH1 signaling both rescues the button defect in Flt4iLECKO mice and accelerates button formation in healthy mice, it is likely that the deletion of Notch1 phenocopies the Flt4iLECKO mice and prevents button formation, but this remains to be tested in future studies.While congenital lymphedema is caused by genetic mutations, acquired lymphedema develops after injury to the lymphatic vasculature.Notch1fl/fl conditional knockout mice to answer this question. Another limitation extends to extrapolating our findings here to the Milroy patients. Here, we are deleting both alleles of the Flt4 gene, leading to a complete loss of protein and signaling, whereas the Milroy patients commonly harbor a point mutation in the kinase domain of Flt4 that acts in a dominant-negative manner.30 Although the patients have a severe loss of VEGFR3 signaling, there is likely some residual signaling remaining. Future studies using the Chy mouse strain will be needed to confirm our findings here regarding defective button junction formation.While we show here that VEGFR3 is needed for postnatal button formation and is dispensable for maintaining buttons, our study has a few limitations. One limitation is that we show that NOTCH1 NICD is capable of rescuing button formation in the absence of VEGFR3, but we have not determined whether NOTCH1 is required for normal button junction formation. We will need to conduct future studies using In summary, our data show that VEGFR3 is required for button junction formation in lymphatic capillaries and interstitial fluid absorption. We were able to rescue this defect by overexpressing NICD1 to enhance NOTCH1 signaling, restoring lymphatic function in these mice. Finally, we show that overexpression of NICD1 can accelerate button junction formation, suggesting that NOTCH1 may be an important inducer of button junction formation developmentally.jscallan@usf.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Josh Scallan  and the Kdrfl/fl strain (Strain#: 018977) were purchased from The Jackson Laboratory.37 Both male and female mice were used on a C57BL/6J background and displayed no sex differences in phenotypes, but individual mice were not sexed. Mice had ad libitum access to food and water. To breed the final mice used in this study, male mice expressing Prox1CreERT2 along with a homozygous floxed gene were crossed with females expressing only the homozygous floxed gene without Prox1CreERT2. One parent expressed a heterozygous Prox1GFP allele or R26-NICD allele. In this manner, littermate controls and knockouts were stained and quantified together but not every pup harbored the Prox1GFP allele. To delete genes at birth, pups were injected subcutaneously with TM (100 μg in5 μL of sunflower oil) from postnatal day P1 and then every other day after until analysis at P21. To delete genes in weanlings, mice were injected intraperitoneally (1 mg in 50 μL of sunflower oil) starting at P21 and then every other day after until analysis at P35. In experiments with the Prox1CreERT2;R26-NICD1 mice, tamoxifen was injected at P1 and P3, as previously published.11 All experiments were performed in accordance with the University of South Florida guidelines and were approved by the institutional IACUC committee.Mouse strains used in this study include FLT4 (shFLT4) (sequence: GAGAGACTTTGAGCAGCCATT)38 for 48 h (VectorBuilder). The virus was removed, and the cells grown for a further 48 h before collection of either protein or RNA.Primary human dermal lymphatic endothelial cells  were cultured on fibronectin-coated plates using EBM-2 media (PromoCell). All hdLECs were used at passage 6–7 for shRNA knockdown. Cells were infected with either a scrambled control (Scr) or an shRNA targeting Mice were anesthetized with ketamine and placed prone on an acrylic board. A 2% solution of Evans blue dye and 2% BSA diluted in sterile saline was freshly prepared prior to the experiment. Approximately 2 μL of the Evans Blue dye was injected into the center edge of the ear using a glass needle attached to a syringe. The glass needle was fabricated by pulling capillary glass on a vertical puller  before grinding the tip to a diameter of approximately 100 μm. Images of the dye-filled lymphatics in the ear were captured using a color camera (Zeiss Axiocam 208 Color) mounted on a dissection microscope (Zeiss Stemi 508).39 Mice were anesthetized with ketamine and placed prone on an acrylic board. A 25 μL glass syringe with a 30-gauge needle was used to inject 2 μL of the dye conjugate  into the mouse ear of P24 mice. Ears were immediately imaged on an Olympus MVX10 fluorescence stereomicroscope outfitted with a Photometrics Evolve 512 Delta EMCCD camera controlled by μManager open source software.Bovine serum albumin (BSA) was conjugated to Alexa 790 and purified through centrifugal molecular weight cutoff filters as previously described.11 Unless otherwise stated, all procedures were performed at 4°C on an orbital shaker . Briefly, tissues were fixed overnight with 2% PFA in PBS and then washed with PBS before permeabilizing for 1 h with PBS +0.3% Triton X-100 (PBST). Tissues were blocked for 2 h with 3% donkey serum in PBST and incubated overnight with primary antibodies diluted in PBST. After five 15-min washes with PBST, tissues were incubated with secondary antibodies diluted in PBST for 2 h on an orbital shaker at room temperature. Following five 15-min washes in PBST, the tissues were incubated with DAPI (Sigma) at room temperature for 5 min and then washed in PBS. Tissues were then mounted onto glass slides  using ProLong Diamond Antifade Mountant (Invitrogen) and stored at 4°C. Details of primary and secondary antibodies are outlined in the resource table. Images were acquired using a 40x water immersion objective on a Leica SP8 confocal microscope and analyzed with FIJI software (NIH). Figures were created using Adobe Photoshop.Whole-mount immunostaining was performed as described previously.7 Buttons were considered short, discrete, unconnected sections of VE-cadherin protein that were perpendicular to the cell membrane. To objectively distinguish between button, intermediate and zipper junctions, we measured and averaged the length of each junction type from 3 samples, allowing us to define a button junction as <4.72 μm in length, intermediate junction as >4.72 μm but <7.85 μm in length, and a zipper junction as >7.85 μm in length. Using these criteria, the total length for each junction type was calculated as a percentage of total junction length.The junction types were quantified using FIJI software from images of lymphatic capillaries. Three types of junctions have been defined – buttons, intermediate, and zipper junctions. We quantified each junction type by measuring junction lengths and then normalizing as a percentage of total length as previously published.Total protein was collected from hdLECs lysed with RIPA buffer  and the protein concentration was measured with a BCA protein assay kit . Gel electrophoresis was performed using the mini gel tank (Invitrogen), the iBlot 2 dry blotting system (Invitrogen), and the iBind automated western system (Invitrogen). Protein was visualized using SuperSignal west pico plus chemiluminescent substrate (ThermoFisher Scientific).Total RNA was collected from hdLECs according to manufacturer’s instructions in the RNeasy plus mini kit (Qiagen). cDNA was synthesized according to manufacturer’s instructions in the Advantage RT-for-PCR kit . The QuantStudio 6 Pro Realtime System (Applied Biosystems) was used to carry out quantitative RT-PCR using Taqman probes (ThermoFisher). The cycle threshold (Ct) value for GAPDH was used to normalize the Ct value for each gene of interest.All junction quantification was analyzed by two-way ANOVA with Sidak’s post hoc test. All qRT-PCR data were analyzed by unpaired Student’s t-test. All graphical data are presented mean ± standard deviation, except for 1