Podosphaera tridactyla species complex is highly variable morphologically and causes powdery mildew on a wide range of Prunus species, including stone fruit. A taxonomic revision of the Po. tridactyla species complex in 2020 identified 12 species, seven of which were newly characterised. In order to clarify which species of this complex are present in Australia, next generation sequencing was used to isolate the fungal ITS+28S and host matK chloroplast gene regions from 56 powdery mildew specimens of stone fruit and ornamental Prunus species accessioned as Po. tridactyla or Oidium sp. in Australian reference collections. The specimens were collected in Australia, Switzerland, Italy and Korea and were collected from 1953 to 2018. Host species were confirmed using matK phylogenetic analysis, which identified that four had been misidentified as Prunus but were actually Malusprunifolia. Podosphaera species were identified using ITS+28S phylogenetic analysis, recognising three Podosphaera species on stone fruit and related ornamental Prunus hosts in Australia. These were Po.pannosa, the rose powdery mildew, and two species in the Po. tridactyla species complex: Po. ampla, which was the predominant species, and a previously unidentified species from peach, which we describe here as Po. cunningtonii.The  Prunus persica), cherries (Pr. avium), apricots (Pr. armeniaca), plums  and almonds (Pr. dulcis) belong to the genus Prunus (Rosaceae), which contains approximately 250 species distributed across temperate regions worldwide [Prunus species, Pr. brachystachya and Pr. turneriana; both are in the subgenus Cerasus and found in tropical rainforests of north east Australia. A further 15 species of Prunus have been introduced to Australia as horticultural crops and for use in gardens [Cerasus (cherries), Padus  and Prunus (plums) and the latter subgenus is further subdivided into seven sections: Amygdalus, Armeniaca, Emplectocladus, Microcerasus, Persicae, Prunocerasus and Prunus [Cerasus has corymbose inflorescences, Padus has racemose inflorescences and Prunus has solitary inflorescences [Stone fruit such as peaches  and rDNA ITS  sequence analyses [Prunus sp., Pr. cerasifera and Pr. armeniaca . Clade 2 contained RFLP Groups 1–4 from Pr. persica, Pr. japonica, Pr. padus, Pr. laurocerasus and Pr. lusitanica . Clade 3 contained RFLP Group 6 from an unknown Prunus sp. and Pr. apetala (Prunus subgenus Cerasus). The clade on hosts from Prunus subgenera Cerasus, Padus and Prunus represented Po. tridactyla in the strict sense, originally described from Pr. padus, while the other two taxa were undescribed species morphologically indistinguishable from Po. tridactyla in the strict sense.In 2005, Cunnington and co-workers investigated genetic variation within analyses . RFLP anPo. tridactyla as a species complex, with no clear morphological delimitation between “typical” collections of Po. tridactyla and deviating forms. They did recognise Podosphaera longiseta as a distinct species within the Po. tridactyla species complex, but concluded that further molecular, biological and morphotaxonomic studies were required to fully recognise species diversity within the species complex. In their taxonomic revision of Erysiphales, Braun and Cook  treated Prunus, examining 30 specimens from 16 hosts and five countries across Asia and Europe. They characterised the Po. tridactyla species complex as comprising 12 species, of which seven were newly described: Po. ampla, Po. pruni-avium, Po. pruni-cerasoidis, Po. prunigena, Po. pruni-japonicae, Po. pruni-lusitanicae and Po. prunina. Additionally belonging to the complex were Po. tridactyla in the strict sense, Po. longiseta and Po. salatai and the two undescribed Podosphaera species from Australia. These two undescribed species formed distinct lineages but could not be described as the authors did not have access to the physical specimens, only DNA sequence data from Cunnington et al. [Prunus, suggesting a degree of coevolution between species of the Po. tridactyla complex and their hosts. The radiation of Prunus subgenera identified by Chin et al. [Po. tridactyla complex species across Eurasia, for example, Po. tridactyla in the strict sense is a European species found on hosts within Prunus subgenus Padus and Po. ampla is of Asian origin, infecting hosts within the Prunus subgenus Prunus [Meeboon et al.  publishen et al. . Addition et al.  identifin et al.  conformss Prunus .Po. tridactyla complex are present in Australia, utilising the next generation sequencing (NGS) methods developed by Smith et al. [Prunus species and closely related ornamental Prunus species in Australia held in Australian plant pathogen reference collections. The objective of the current study was to clarify which species of the h et al. , based oPodosphaera tridactyla or Oidium sp. on Prunus hosts were obtained from the three major Australian plant pathogen reference collections (Queensland Plant Pathogen Herbarium (BRIP—two collections), New South Wales Plant Pathology Biosecurity Collections (DAR—32 collections) and Victorian Plant Pathogen Herbarium (VPRI—125 collections)). Collections in BRIP and DAR were all from Australia, while those in VPRI were from Australia, Switzerland, Italy and Korea. All collections were inspected for DNA extraction suitability as described by Smith et al. [All powdery mildew collections identified as Powdery mildew conidia, mycelia and chasmothecia were collected from the specimens by using a 6 mm leaf punch or scraping with a blade. Sampling was completed under clean room conditions to minimise contamination with modern DNA.2O) 20 mL and acid fuchsin 0.1 g; this was adapted from the Johnson and Booth [The VPRI specimens collected between 1993 and 1995 were morphologically examined while fresh at the time of collection. The hyphae, conidiophores and conidia were collected off the leaf surface with clear cellotape, which was then mounted on a microscope slide with lactofuchsin mountant and observed using light microscopy. Lactofuchsin preparation was as follows: lactic acid 20 g, glycerol 40 g, water , conidial chain edge line, conidial size and shape, presence or absence of fibrosin bodies and germination characters. ® Forensic DNA protocol [® electrophoresis .DNA was extracted following the E.Z.N.A.protocol . The DNALibrary preparation was as described in Smith et al. . LibrariPodosphaera DNA and plant host DNA, as well as DNA from microbes present on the leaf surface at the time of collection. Therefore, the filtered, high-quality (HQ) reads were mapped as paired-end reads to Podosphaera and plant host reference sequences. For Podosphaera, the reference sequences used were the internal transcribed spacer ITS (ITS1-5.8S-ITS2) and 28S rDNA regions of Po. ampla (GenBank accession MK530453), Po. cerasi (KX826855), Po. clandestina (KY660805), Po. leucotricha (KX842350), Po. longiseta (MK530459), Po. pannosa (KX842349), Po. prunigena (AB936275), Po. prunina (MK530442), Po. pruni-avium , Po. pruni-cerasoides (MK530448), Po. pruni-japonicae (MK530455), Po. pruni-lusitanica (KP641993), Po. salatai (AB525929), Po. sp (AY833653) and Po. tridactyla s. str. (MK530462). The plant host was identified by mapping to matK chloroplast plastid regions. Reference sequences were those identified by Chin et al. [matK accessions from Chin et al. [Pr. armeniaca (HQ235100), Pr. cerasifera (HQ619834), Prunus domestica (HQ235146), Pr. laurocerasus (HQ235181), Pr. mahaleb (HQ235184), Pr. padus (HQ235216), Pr. persica (HQ235409), Pr. salicina (HQ235252), and the others from GenBank were Pr. yedoensis (GQ248191) and Malus prunifolia (JQ391019). The sequence mapping was performed by the program Nuclear, generating reference-initiated sequence alignments to be viewed in Vision software . In Vision, ITS+28S and matK sequences were edited to incorporate single nucleotide polymorphisms (SNPs) and indels which related to each individual specimen’s sequence data. A mapping threshold was determined by a minimum of 5x coverage of the complete reference sequence and sequence files which did not meet this criterion were excluded. Mapping success was determined from the Vision images of each sequence file by calculating the total number of aligned DNA sequence reads and converting it into a percentage from the total number of HQ reads. The mapped ITS+28S and matK sequences generated for this study were exported from Vision for identification to species by BLASTn and phylogenetic analysis.Sequence reads were assigned to each sample based on their indices. Raw DNA-Seq files were filtered using the Nuclear program to trim adapters and retain only the sequences above the cut-off value of 100 bp read length and 20 mismatches. The library preparations were expected to contain Po. tridactyla. The first data set  sequences from the Po. tridactyla complex  as the outgroup taxon, in line with Meeboon et al. [matK sequences included sequences from Prunus species subgenera Cerasus, Padus and Prunus obtained from Chin et al. [Oemleria cerasiformis (matK AF288110). Alignments were generated in Geneious Prime using the Muscle 3.8.425 [Based on preliminary phylogenetic analysis, two data sets were generated for the sequences originally identified as data set  was cons complex . Both da studies ,16,17 ann et al.  and the  3.8.425 ,19 align 3.8.425  with the 3.8.425  using twPo. ampla and sister taxa and (2) species closely related to Po. pruni-avium. For each set of sequences, outgroup sequences were removed, and the sequences of interest were realigned. Masking of the amended alignments by removing sites containing identical bases, leaving only sites with variable bases (including gaps), was performed by the Mask Alignment tool in Geneious Prime. In addition, positions where only one sequence within a species varied from the consensus were removed manually in Geneious Prime. Base position numbers of the variable sites were manually edited in Microsoft PowerPoint to reflect base positions in the original alignment.To identify characteristic bases for sets of closely related species, summaries of variable base positions were generated for (1) Podosphaera) ITS+28S region and host plant chloroplast gene matK. The exceptions were DAR 35281 and DAR 64667, which failed to meet the mapping threshold. The ITS+28S and matK sequences generated from this study were confirmed as belonging to Podosphaera and Prunus, respectively, using BLASTn. The BLASTn analysis of the powdery mildews detected six collections which did not match with species of the Po. tridactyla complex and matched to either Po. pannosa or Po. leucotricha. These six sequences were placed in a data set with other sequences identified at Po. leucotricha and Po. pannosa as well as closely related Podosphaera species, as they are not part of the Po. tridactyla species complex. The remaining ITS+28S sequences returned BLASTn percentage identities of at least 98% and up to 100%  with members of the Po. tridactyla species complex. DNA from 56 of the 58 collections were successfully mapped to reference sequences of the powdery mildew  and DAR 12478  Another well-supported clade  consisted of seven sequences identified as Po. leucotricha along with sequences from VPRI 12495, 18514, 18452 and 20687   .Po. tridactyla species complex: Po. ampla on Prunus subgenus Prunus hosts and a previously undescribed Podosphaera species on Prunus persica and Pr. mahaleb . Within this clade, there was a moderately supported BI 0.85 subclade containing the Po. ampla reference sequences from Germany and nine sequences from Australian collections , Po. prunina , Po. pruni-japonicae (VPRI 20233 and 20490 South Korea) and Po. pruni-avium  . A welllections . Within zerland)   but included three variants at positions 545 (A instead of G), 546 (T instead of A) and 550 (A instead of G). A summary of variable bases for the d of a C . Sequencd of a C . SequencPo. ampla and the unknown species of Podosphaera). Support values for the Po. ampla clade, including the Australian sequences, were as follows: ITS  and 28S  , including 134 sequences covering 486 base pairs, and of 28S (large subunit) including 73 sequences of 808 base pairs, formed the same overall structure regarding all major clades. All Australian sequences generated from this study fell into two well-supported clades  . In the d 28S BS 0, BI 1  published by Cunnington et al. [Podosphaera sp. by Meeboon et al. [Po. tridactyla complex. In the ITS+28S phylogeny, support for this new Podosphaera species is high (BS 84 and BI 1)  . The indnd BI 1) . A summaaponicae . The newPrunus subgenera compared to the hosts detected by Meeboon et al. [Po. pruni-avium, Po. pruni-japonicae and Po. prunigena, were all originally described from Prunus hosts within the subgenus Cerasus, but in the ITS+28S phylogeny, the clades for these species included VPRI 22159, VPRI 20490, VPRI 20233 and VPRI 20491 on Pr. padus, which is in subgenus Padus. The Podosphaera pruni-japonicae clade in the ITS tree also included sequences from fungi collections on Prunus davidiana (subgenus Prunus) , also included a sequence from a collection on Pr. mahaleb (subgenus Cerasus). Both Po. prunina and Po. ampla clades contained powdery mildew sequences generated from Prunus hosts within subgenus Prunus in all three phylogenetic analyses.The ITS+28S phylogeny of the fungi showed several sequences from hosts belonging to different n et al.   . The ITS Cerasus . The claPrunus species originally listed as the plant hosts for Australian Po. tridactyla complex collections . The plant host species were analysed in a matK phylogenetic analysis containing 133 sequences of 1288 base pairs, including representatives of the three subgenera of Prunus (There were nine different f Prunus . Prunus subgenera Cerasus (clade 1), Padus (clade 2) and Prunus (clade 3). The matK sequences for DAR 28963, VPRI 20491 and VPRI 20231 fell into clades with sequences for Pr. mahaleb and Pr. yedoensis belonging to subgenus Cerasus within the Padus clade, VPRI 22156 and BRIP 8323 formed a well-supported clade  with sequences identified as Pr. laurocerasus. This is a host reidentification for BRIP 8323 which had Pr. persica listed on the specimen. Three VPRI sequences, 20490, 20233 and 22159, formed a clade with no BS support and BI support below the 0.85 threshold with Pr. padus sequences and close relatives such as Pr. grayana and Pr. virginiana. Within the subgenus Prunus, section Amygdalus formed a clade that was only supported by BI (0.91) containing sequences identified as Pr. persica along with sequences VPRI 19591, 20705, 20706, 22232 and 22233  along with DAR 28962 and DAR 71638 . In section Prunus, the sequences identified as Pr. armeniaca formed a clade including eleven VPRI and DAR specimens identified as Pr. armeniaca with BI support of 0.95. The remaining VPRI sequences  fell within a clade comprising sequences identified as Eurasian plums of sect. Prunus with high BI support (0.92) and no BS support. The Eurasian plums included Pr. domestica, Pr. cerasifera and Pr. salicina. There are minimal differences between these species within the matK gene region and therefore, for the purpose of identification, this group is referred to as the Pr. domestica group which includes the previously mentioned Prunus species as well as Pr. brigantina, Pr. consociiflora, Pr. simonii and Pr. spinosa. Additionally included in the clade of Eurasian plums were sequences from BRIP 15118 and VPRI 19868, which were both previously misidentified as Pr. persica  and the associated powdery mildews were all re-identified as Po. leucotricha.There were four VPRI specimens, VPRI 12495, 18452, 18514 and 20687, which are labelled with plant hosts listed as Po. pruni-avium and Po. pruni-japonicae. No sexual morphs were found among material of this species. The morphologies of the sister taxa barely differ from Po. tridactyla in the strict sense and the asexual characters of this undescribed species on Prunus persica and Pr. mahaleb are the shorter conidiophore foot cells, crenate conidial chains and smaller conidia when compared to characters of the asexual morph of Po. tridactyla, which has long foot cells and ellipsoid(-doliform) conidia catenescent with crenate edge line . Etymology: the epithet commemorates Dr. James Cunnington, the first person to conduct molecular examination of this species.Mycelium hyphae branched, thin walled, sinuous. Hyphal appressoria nipple shaped. Conidiophores straight, basal septum slightly displaced at junction, foot cells (30–) 43–60 × 8–11 µm, succeeded by 2–3 following cells, forming conidia in chains with crenate edge lines. Conidia ovoid(-doliform), 23–25 × 12–15 µm, fibrosin bodies present in water mounts, lacking in lactic acid mounts. Germination on host tissue at 24 h, producing single oblong-clavate, simple germ tubes 20–30 × 5–6 μm; at 48 h, producing 1–2 simple germ tubes 25–35 μm. No sexual morph observed. Natural distribution: currently unknown as isolates were collected in quarantine glasshouses in Australia. Additional collections examined: AUSTRALIA, VICTORIA: Knoxfield, isolated from Pr. persica, 7 March 1994, A. Sivapalan VPRI 19,868; isolated from Pr. persica, 27 September 1995, V. Beilharz VPRI 20,705; isolated from Pr. persica, 27 September 1995, V. Beilharz VPRI 20706; NEW SOUTH WALES: Rydalmere, isolated from Pr. persica, 3 June 1977, L. Penrose and J. Walker DAR 28,962; isolated from Pr. mahaleb, 3 June 1977, L. Penrose and J. Walker DAR 28,963; isolated from Pr. persica, 22 January 1996, G. Stovold DAR 71638 .Po. tridactyla and Oidium sp. on Prunus hosts and, through phylogenetic analysis, found seven different powdery mildew species and corrected misidentified plant hosts of eight specimens. The main species present on Prunus in Australia was Po. ampla and we also found that Australian collections of Po. pannosa and Po. leucotricha had been misidentified as Po. tridactyla and Oidium sp. in two reference collections. Finally, we characterised a new species in the Po. tridactyla complex which we name Po. cunningtonii from Pr. persica and Pr. mahaleb plant hosts. This study re-evaluated reference collection specimens of Po. ampla by Meeboon et al. [Po. ampla had a one base pair difference compared to the Po. ampla sequences analysed in previous studies. The revision of the Po. tridactyla species complex by Meeboon et al. [Po. ampla holotype MK530453 but were still included under Po. ampla. Therefore, most of the Australian powdery mildew fungi on stone fruit  formerly identified as Po. tridactyla are in fact Po. ampla.The phylogenetic analysis based on sequences derived from NGS shows that 37 of the 56 specimens fell within a clade alongside sequences identified as n et al. . Most Aun et al.  includedPo. ampla and fell within a well-supported clade with Po. ampla sequences analysed by Meeboon et al. [Podosphaera tridactyla sequences AF154321 and AY883656 generated by Cunnington et al. [Po. ampla sequences, the only differences being that the sequence lengths were significantly shorter than the ITS+28S sequences which were generated by this study and Meeboon et al. [Po. ampla.The ITS phylogeny and 28S phylogenies also confirmed that most of the Australian sequences were n et al.   and two specimens from BRIP and DAR were Po. pannosa, which is more commonly seen on roses. The remaining Po. tridactyla complex species were identified from collections made in Switzerland and South Korea but held at VPRI.The remaining species which were identified from the Australian reference collections were: Po. ampla, six of the sequences newly generated by NGS were of an unknown species which did not match any identified sequences on GenBank. Cunnington et al. [Po. tridactyla complex. In the Cunnington et al. [Podosphaera on Pr. japonica, which was renamed Po. pruni-japonicae by Meeboon et al. [Po. tridactyla not on Prunus subgenus Prunus. In the Meeboon et al. [Po. pruni-japonicae and Po. pruni-avium. The results of the present phylogenetic study align with both Cunnington et al. [Po. cunningtonii including additional sequences generated in this study form a well-supported clade which is sister to a clade comprising Po. pruni-japonica and Po. pruni-avium.While most Australian specimens were n et al.  and Meebn et al.  both stan et al.  study, tn et al. , within n et al.  study, tn et al.  and Meebn et al. , where tPodosphaera species on Prunus persica returned 26 sequences of two powdery mildew species, Po. pannosa and Po. leucotricha (peach rusty spot), which are both found on peach [Pr. persica included in the host ranges, these are Po. tridactyla in the strict sense, Po. prunicola and Po. pannosa, which are quite separate to Po. cunningtonii in the phylogeny. A GenBank search for sequences of on peach ,23,24. Hon peach  list thrPo. cunningtonii in Victoria (mid-1990s), it was assumed that the quarantine glasshouse-grown Pr. persica had been infected by powdery mildew blowing into the glasshouse (Cunnington 2020 pers. comm.). However, in this study, we identified the same powdery mildew fungi on three specimens from New South Wales, two of which (DAR 28962 and DAR 28963) were also collected in quarantine glasshouses. The two specimens from quarantine glasshouses in N.S.W. are on two different hosts—Pr. persica (subgenus Prunus) and Pr. mahaleb (subgenus Cerasus). Notes in the specimen packet reported that the Pr. mahaleb seedlings were growing next to heavily powdery mildew-infected Pr. persica seedlings in the same glasshouse. The determiner, Dr. John Walker, noted at the time of collection (1977), “mildew development very light and feel that this is an adventitious development of mildew on this host from the heavily mildewed peach seedlings growing nearby”. We suggest that the primary host of this new powdery mildew is Pr. persica but under glasshouse conditions it can infect other Prunus species from different subgenera as accidental hosts. At the time of the first collections of Po. cunningtonii, from N.S.W. (DAR 71638), was collected from Pr. persica on a private property in 1996, in a locality with several orchards that grew peach and other stone fruit in the near vicinity. This is the only report of Po. cunningtonii from outside of quarantine glasshouses and therefore we cannot confirm if it is established in Australia. Two specimens of powdery mildew on Pr. persica collected in 1991 from a quarantine facility in Alice Springs, Northern Territory, are lodged in the Northern Territory Plant Pathogen Collection (DNAP). Unfortunately, we were unaware of them at the time of commencement of the present study, so they were not included. At the time of collection, they were identified morphologically as Po. pannosa by determiner Dr. Jose Liberato. The two native Prunus species, Pr. brachystachya and Pr. turneriana, are only found in tropical rainforests in the far north of Queensland of Australia. Horticultural Prunus species, including stone fruit, are grown in the temperate regions of Australia further south [Prunus powdery mildew fungus onto commercial stone fruit crops and to date there are no records of powdery mildew fungi infecting these native hosts. It is highly unlikely that Po. cunningtonii has a native area of distribution in the natural distribution of Pr. persica which originates from Northwest China. New peach varieties are imported into Australia as budwood and grown under quarantine prior to release. Potential explanations as to why Po. cunningtonii has not been observed more widely in Australia could be that infected plants would have not been released from quarantine. Another reason could be that the local climate in peach-growing regions in Australia is not climatically suitable for Po. cunningtonii development, whereas the quarantine glasshouse conditions provided an artificial environment for Po. cunningtonii growth [A third specimen of er south ,6. Geogri growth ,26. Australia is believed to be a continent without native powdery mildew fungi, with accidental human-assisted powdery mildew introductions occurring post-European settlement on non-native agricultural, horticultural, ornamental and pasture plant species . FurtherPo. tridactyla specimens and we also identified and described a previously unrecorded powdery mildew species, Po. cunningtonii. Outcomes such as these highlight the need to re-evaluate important plant pathogen groups held in reference collections, in particular unculturable pathogens and complexes of cryptic species [Over the last 50 years, there have been changes in the way that specimens are collected, preserved, studied and recorded in reference collections . These c species . Austral species . Po. ampla, is the dominant powdery mildew infecting stone fruit in Australia. In addition, the phylogenetic study confirmed the presence of Po. pannosa in Australia and allowed delimitation of a new powdery mildew species within the Po. tridactyla species complex, Po. cunningtonii on Pr. persica, for which we provide a morphological and molecular description. This study resolved powdery mildew species but also identified Prunus plant hosts and reclassified previously misidentified or incompletely identified hosts of specimens held in Australian plant pathogen reference collections. The information generated in this study will be used to update BRIP, DAR and VPRI powdery mildew specimens to provide accurate data for Australian biosecurity agencies. Through NGS applications, re-examination of selected powdery mildew specimens from Australian plant pathogen reference collections has demonstrated that one species,


Antigen recognition of allo-peptides and HLA molecules leads to the activation of donor-reactive T-cells following transplantation, potentially causing T-cell-mediated rejection (TCMR). Sequencing of the T-cell receptor (TCR) repertoire can be used to track the donor-reactive repertoire in blood and tissue of patients after kidney transplantation.In this prospective cohort study, 117 non-sensitized kidney transplant recipients with anti-CD25 induction were included. Peripheral mononuclear cells (PBMCs) were sampled pre-transplant and at the time of protocol or indication biopsies together with graft tissue. Next-generation sequencing (NGS) of the CDR3 region of the TCRbeta chain was performed after donor stimulation in mixed lymphocyte reactions to define the donor-reactive TCR repertoire. Blood and tissue of six patients experiencing a TCMR and six patients without rejection on protocol biopsies were interrogated for these TCRs. To elucidate common features of T-cell clonotypes, a network analysis of the TCR repertoires was performed.+), p = 0.52 (CD8+)]. Graft-infiltrating T-cells showed an up to six-fold increase of donor-reactive T-cell clonotypes compared to the blood at the same time (3.7 vs. 0.6% and 2.4 vs. 1.5%), but the infiltrating TCR repertoire was not reflected by the composition of the circulating TCR repertoire despite some overlap. Network analysis showed a distinct segregation of the donor-reactive repertoire with higher modularity than the overall TCR repertoire in the blood. These findings indicate an unchoreographed process of diverse T-cell clones directed against numerous non-self antigens found in the allograft.After transplantation, the frequency of circulating donor-reactive CD4 T-cells increased significantly from 0.86 ± 0.40% to 2.06 ± 0.40% of all CD4 cells . The number of circulating donor-reactive CD4 clonotypes increased from 0.72 ± 0.33% to 1.89 ± 0.33% . No difference in the percentage of donor-reactive T-cells in the circulation at transplant biopsy was found between subjects experiencing a TCMR and the control group [p = 0.64 .Clinicaltrials.gov: NCT: 03422224 ( Despite the improvement of immunosuppressive therapies in kidney transplantation, early acute T-cell-mediated rejection (TCMR) occurs in roughly 10% of patients and may impact graft function and survival if not detected early and treated appropriately . The patThis recognition of non-self peptides and HLA molecules leads to an alloresponse, and recently we showed that also mismatched non-HLA antigens between donor and recipients induce an immune response that is associated with a reduced graft survival .In the setting of solid organ transplantation, recent papers described the clonality and diversity of the allo-TCR repertoire in the circulation after lymphodepletional induction therapy with anti-thymocyte globulin (ATG). DeWolf et al. showed that in healthy controls, roughly only 0.4 to maximal 10% of the whole TCR repertoire in blood is donor-reactive .Accordingly, even in severe viral infections such as BK, CMV, or also tuberculosis, investigators showed that only a small fraction of each individual TCR repertoire undergoes viral or pathogen-specific expansion, but the majority of the repertoire remains unchanged –10. In tIn a previous study involving a small number of kidney transplant recipients, those receiving conventional immuno-suppression demonstrated a similar increase in circulating donor-reactive T cells defined by mixed lymphocyte reaction (MLR) sequencing, regardless of whether or not rejection occurred . What reMorris and colleagues recently tracked donor-reactive T-cells in a cohort of four simultaneous kidney and bone marrow transplant recipients. Three of these patients developed operational tolerance, and TCR sequencing of peripheral T-cells suggested that long-term tolerance reflected clonal deletion of donor-reactive T-cells. The one patient with TCMR following immunosuppression withdrawal did not show evidence of deletion, in contrast to tolerant patients .In a recent study focusing on the B-cell repertoire after transplantation, a difference in clonal expansion between patients with rejection and no rejection and the usage of specific immunoglobulin gene segments in those patients was shown . ClusterGiven the uncertainty of TCR repertoire kinetics in TCMR, we sought to prospectively elucidate the dynamics of clonality and diversity in blood and tissue of patients after kidney transplantation.The detailed design of this prospective observational cohort study may be found elsewhere . In brieA STROBE chart of included patients is provided in in vitro experiments, but one had to be excluded due to insufficient T-cell numbers found in the pre-transplant blood and two were lost due to sorting errors. Overall, the MLR, FACS sorting, and sequencing were successfully performed for three patients with histologically proven pure acute TCMR and three patients with a borderline rejection. The TCRs were compared to a control group of six patients without histopathological signs of alloimmune response in kidney biopsies after transplantation. Biopsy specimens for TCR sequencing were available for two rejecting patients and compared to two biopsy specimens of the control group. The control group was selected based on the time of biopsy. Detailed patient characteristics are listed in 5 carboxyfluorescein succinimidyl ester (CFSE)-labeled (Invitrogen cat. #C34554) transplant-recipient cells and 2 × 105 VPD-labeled (BD horizon cat. #562158) irradiated donor cells in each well of a 96-well plate. MLR cultures were incubated at 37°C for 6 days and were sorted on a FACS Aria II high-speed cell sorter for CD4+ CFSElow and CD8+ CFSElow T-cells   , 21. UnsA historical cohort of seven kidney transplant recipients experiencing a cellular rejection episode was used to confirm findings in our cohort. Institutional ethics committee approval ) was obtained for all aspects of the study, and all study participants were included after signed informed consent prior to transplantation. Patient characteristics of the historical cohort are shown in RNA isolation, next-generation sequencing (NGS) library preparation, sequencing, and bioinformatics analysis were previously described . In brieTotal RNA was successfully isolated of all biopsy samples stored in Tissue-Plus OCT compound and subjected to library preparation except for one control due to mainly muscle and fat in the biopsy. For RNA from tissue specimens from the control group, no TCR amplicon could be obtained. This was concordant with histological absence of mononuclear cell infiltrates. TCR libraries were sequenced on an Illumina NextSeq 500, and reads were stored in separate fastq files per index. The MIGEC pipeline was used for further demultiplexing and unique molecular identifier (UMI)-guided consensus sequence assembly . ResultiFor each TCR β chain, the following information was retained for further analysis: CDR3 sequence (amino acid and nucleotide translation), cell counts, and frequency of clones with regard to the nucleotide sequence, V gene, D gene, and J gene. Ambiguous clonotypes with regard to its phenotype were assigned CD4 or CD8 according to the subset in which their maximum frequency across timepoints doubles the other. The remaining clonotypes were dropped due to their questionable affiliation. Clonotypes were defined as donor-reactive if they fulfilled a fold change equal or greater than 5 with regard to abundance between the stimulated and the unstimulated pre-transplant sample. The datasets for this study can be found in the European Genome-Phenome Archive .To correct for possible bias due to differences in the number of captured T-cells, the assessment of the frequency distribution of donor-reactive clonotypes in each repertoire was performed based on the mean value from 1,000 estimates, each obtained from random downsampling of the respective sample to the total number of T-cells in the overall repertoire with the lowest read count . The aveobsH).The diversity of TCR clones in circulation and tissue was quantified by R20 and clonality. R20 is defined as the fraction of the most frequent clonotypes within the repertoire which together make up 20% of the entire repertoire. Hence, a relatively small R20 value indicates immunodominance of a subset of clonotypes, whereas an inflated number points toward the lack thereof. Clonality (C) provides a measure of normalized diversity and is derived from Shannon’s entropy of a sample .The similarity overlap of inter-sample repertoires was assessed by the Jensen–Shannon divergence (JSD) of the top 1,000 clones, a quantitative measure of divergence between two probability distributions .The function is bounded by 0 and 1, where a JSD equal to 1 would represent two fully distinct clonal distributions and 0 identical distributions.TCR networks were constructed as undirected and unweighted graphs with nodes corresponding to unique CDR3 β amino acid sequences connected by edges if a similarity condition based on the overlap of amino acid is fulfilled. The selected measure of node similarity was the Levenshtein distance, a string metric quantifying the difference between two CDR3 β amino acid sequences by counting the number of edits  required to convert one sequence into the other –30. Non-The numbers of detected TCR clonotypes pre-transplant were comparable to those after transplant .Among all unique complementarity-determining region 3 (CDR 3) sequences found in the patients’ blood stream pre-transplant, the percentage of donor-reactive T-cell clonotypes ranged from 0.34 to 1.56% for CD4 and from 0.16% to 2.73% for CD8 clonotypes. Looking at the abundance of donor-reactive T-cells in the circulation, regarding their frequency, revealed that 0.43% to 1.92% of all CD4 and 0.15 to 1.49% of all CD8 cells were donor directed Figure 3vs. rejection group was 6 to 141 days (median 61 days) vs. 7 to 251 days (median 94 days) after transplantation, and the absolute number and percentage of donor-reactive clones in CD4 and CD8 T-cells increased in almost all subjects compared to pre-transplant in the peripheral blood stream  and p = 0.52 (CD8+)], and overall the increase in donor-reactive clones was more pronounced in the CD4 fraction compared to the CD8 alloclones.The timepoint of biopsy for the control The diversity of pre-transplant repertoires measured by clonality ranged from 0.018 to 0.13 for CD4 and 0.13 to 0.5 for CD8 T-cells. The clonality of post-transplant samples showed comparable values to pre-transplant values, as shown in We quantified if the significant increase in alloclones was reflected by overall turnover in the TCR repertoire pre- and post-transplant, which would be indicated by a high Jensen–Shannon divergence (JSD). However, JSD values were low, indicating that there was little overall repertoire change in the sampling period and no statistical difference was seen in rejecting patients and the control group , and the R20 was 0.013 vs. 0.019   Figure 6The individual-specific networks for the donor-reactive immune repertoires showed a significantly higher modularity than the networks of the pre-transplant  and post-transplant repertoires , suggesting a closer connectivity within the formed clusters Figure 7In the individual-specific networks of the tissue repertoire, 9.3% and 22.5% of all donor-reactive clones found in the tissue were included in the respective individual-specific immune networks. Network analysis revealed that the most centrally connected clonotype in both of the individual-specific networks was donor-reactive determined by betweenness centrality (measure of node influence) and degree (measure of the connectivity between the nodes) .Institutional ethics committee approval ) from the Ethics Committee of the Medical University of Vienna was obtained for all aspects of the study and all study participants were included after signed informed consent prior to transplantation.in vitro experiments, and data analysis. TW, MS, JBH, RR-S, HR, and KJ are responsible for the critical revision of the manuscript. AH, MG, SS, SW, JV, and AK are responsible for the statistics, bioinformatic analysis, and revision of the manuscript. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.RO, RR-S, and JW were responsible for the conception, design, financial support, critical revision, and final approval of the manuscript. RO, CA, and AH were responsible for the manuscript writing. KJ, KH, AH, JW, LP, GG, ME, ART, HR, and CA performed the sample collection, https://www.oenb.at). The funding body had no influence on the design, collection, analysis, and interpretation of data and writing the manuscript.The study was founded by a peer-reviewed funding by the Scientific Funds of the Austrian National Bank-OeNb project number 17289 (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Pulmonary ossifications have often been regarded as rare, post-mortem findings without any clinical significance. We have investigated the occurrence of pulmonary ossifications in patients undergoing thoracic procedures, and how this may affect the differential diagnosis of solitary pulmonary nodules. In addition, we have performed a literature search on the occurrence and possible pathogenesis of these ossifications. From January 2008 until August 2019, we identified pulmonary ossifications in 34 patients who underwent elective pulmonary surgery. Pre-operative imaging was unable to differentiate these ossifications from solid tumors. A definitive diagnosis was made by an experienced pathologist . The PubMed database was researched in December 2019 with the search terms “pulmonary ossifications”; “heterotopic ossifications”; and “solitary pulmonary nodule”. In total, 27 patients were male, with a mean age of 63 ± 12 years (age 41 to 82 on diagnosis). All lesions were identified on thoracic CT and marked for resection by a multidisciplinary team. A total of 17 patients were diagnosed with malignancy concurrent with ossifications. There was a clear predilection for the right lower lobe  and most ossifications had a nodular form (70.6%). We could not identify a clear association with any other pathology, either cancerous or non-cancerous in origin. Oncologic or pulmonary comorbidities did not influence patient survival. Pulmonary ossifications are not as seldom as thought and are not just a curiosity finding by pathologists. These formations may be mistaken for a malignant space-occupying lesion, both pre-and perioperatively, as they are indistinguishable in imaging. We propose these ossifications as an underestimated addition to the differential diagnosis of a solitary pulmonary nodule. Mature bone tissue in the alveolar or interstitial spaces is a remarkable finding that was first described in an obduction report by the anatomist Herbert Von Luschka in 1856 [Ossification is defined as deposits of calcium in a collagen matrix in the presence of osteoblastic cells. Two distinct types of PO are distinguished in literature: nodular PO and dendriform PO. Nodular PO are secluded to the alveolar space and are often round. Dendriform PO branch through the interstitium of the septa in a tree-like coral pattern. Sometimes, the bone tissue has matured to encase osteoblasts, osteoclasts, and bone marrow with fatty cells and haematopoetic stem cell tissue . Even wiThere is not much indication that genetic anomalies are responsible for the development of PO, although there has been an occasional finding of familial clustering in a father and son . DeletioIn literature, dendriform ossifications are associated with chronic lung injury and inflammation and nodular ossifications are more often found in patients with passive congestion due to chronic heart failure. This may explain why PO are more often found in the lower lobes, as blood accumulates there in patients with insufficient cardiac function. The subsequent extravasation and degradation of red blood cells result in hemosiderin deposits. These react with calcium that is released by injured tissue to form precipitating salts. The process is further propagated by phospholipids released from injured cell walls that degrade into fatty acids which may also bind free calcium. This ultimately results in the deposition of a calcified matrix in or around the alveoli. Long term lung injury appears to cause metaplasia of fibroblasts into osteoblasts. Two important enzymes activated by lung injury and inducing osteoblast activity are alkaline phosphatase (AP) and transforming growth factor-beta (TGF-β). AP is an enzyme that is abundantly present on type II pneumocytes and catalyzes the production of free phosphate which increases the calcium-phosphate product . TGF-β fWe conducted a retrospective study to identify any cases of PO at the Antwerp University Hospital, a tertiary care center in Belgium. From January 2008 until August 2019, pathologic specimens from all patients undergoing thoracic surgery were examined for the presence of pulmonary ossifications. We collaborated closely with our center’s pathologists dedicated to thoracic pathology  to identify any PO and determine whether their growth pattern could be considered as either nodular (NPO), or dendriform (DPO), or both. All specimens were formalin-fixed and stained with hematoxylin-eosin for subsequent light microscopy. Nodular calcifications without any evidence of ossifications  were omitted from this series. All ossifications lesions were previously identified on thoracic computed tomography (CT) and considered for resection by a multidisciplinary oncologic team. A literature search was conducted through the PubMed database in December 2019 with the search terms “pulmonary ossifications”; “heterotopic ossifications”; and “solitary pulmonary nodule”.A total of 34 cases were identified from the period 2008–2019, with an average incidence of 1.1% to a total of 3122 thoracotomies or thoracoscopies performed at our center during this period. This equated to three cases per year.Most patients were male  with a mean age of 63 years upon diagnosis (range 41 to 82). Nine patients (26.5%) had a history of obstructive lung diseases such as asthma or chronic obstructive pulmonary disease (COPD), and only three patients (8.8%) suffered from interstitial lung fibrotic disease. Two patients (5.88%) were diagnosed with congestive heart failure. Suspected malignancy was, in all cases, the reason to perform surgery. This oncologic diagnosis was confirmed in 19 patients (55.9% of cases). We could not demonstrate any correlation between the imaging and operative findings. Due to their small size, not all pathologically proven ossifications could be visualized on chest CT scans. A representative image is presented in We observed a clear predilection for the lower lobes , and most ossifications were found in the right lung  . We idenp = 0.013) between the presence of bone marrow and arterial hypertension, and elder age . There were no significant correlations between the location of the tumor and the presence of marrow or the difference between nodular or dendriform PO.A Pearson correlation test was performed to determine correlations between parameters. We observed moderate correlations  for oncologic patients was 204.7 weeks, 95% confidence interval (CI) 118.9–290.6 weeks; for non-oncologic patients MST was 125.8 weeks, 95% CI 0–293.6 weeks; for patients with pulmonary disease MST was 158.3 weeks, 95% CI 17.0–299.5 weeks; for patients without pulmonary disease 185.4 weeks, 95% CI 17.0–299.5 weeks.A Kaplan–Meier analysis with Log rank test was performed to determine whether there were any differences in survival time between patients where PO were found either with and without malignancy, or with and without any pulmonary comorbidity . The surIn this paper, we describe 34 cases in total of PO, confirmed after a histopathological analysis, in a retrospective case series that spans eleven years. Originally defined as a post-mortem diagnosis and a rather seldom finding, we argue that PO are truly more common than often thought and deserve to be admitted to the list of possible differential diagnoses for a solitary pulmonary nodule. More awareness of this entity by radiologists, pulmonologists, surgeons, and pathologists may lead to a better understanding of PO and, possibly, an increased prevalence in pre- and postoperative reports.We found that PO most often occur in elder patients and mostly in men. Although the past years have seen an increase in papers reporting on PO, the diagnosis is seldom made following surgery, and most papers focus on post-mortem observations of this condition . Final pDifferentiating between nodular and dendriform types of PO seems a bit arbitrary. We could not find any clear association with a clinical disease, of which congestive heart failure and pulmonary fibrosis are most commonly found to be associated with PO in literature. The presence of PO does not influence patient survival when suffering from any lung disease or oncologic disease, as demonstrated through the calculated survival functions in Furthermore, we confirmed that the nodular form is more abundantly present. When considering the differential diagnosis, dendriform PO may especially be confused with disorders associated with considerable morbidity and mortality such as bronchiectasis, interstitial fibrosis, and even lymphangitic metastasis .We could not find any association between the presence of bone marrow tissue and the differentiation between either NPO or DPO, although the literature suggests that DPO are more commonly associated with marrow formation . The mecA predilection for the lower lobes was found in our study group, which is confirmed by earlier reports . The basAlthough PO are sometimes considered a post-mortem rarity, their clinical implications are clear. We advocate for an increased awareness of this curious finding, from the pre-operative diagnosis by a radiologist who may order a high-resolution CT to differentiate PO from other possibly more severe pathologies such as a metastasis, to the thoracic surgeon who suddenly feels an unexpected nodule during thoracotomy, and finally the pathologist encountering bone formation where least expected. With a population that continues to grow older with both cardiac and pulmonary chronic diseases, we can expect an increase in these incidental findings.To conclude, we reported a case series of 34 patients with different forms of pulmonary ossification. Although the importance of the antemortem diagnosis of PO is underscored in literature, there is certainly a need for clinicians to be aware of this finding. We did not observe any specific correlated pathologies, either cardiac or pulmonary, with any form of ossification. With an increasing population of elderly patients combatting chronic diseases, we may certainly expect a further and steady increase in the diagnosis of these curious findings. Alertness of clinicians in different aspects of medicine to include PO in the differential diagnosis of pulmonary ossifications may result in a further augmentation in diagnoses. Therefore, we propose PO as a valuable addition to the list of differential diagnoses of an SPN.


The previous studies have showed that serum retinol binding protein 4 (RBP4) levels increase in metabolic disorders which are closely associated with cardiovascular diseases (CVD). However, the human studies investigating the role of RBP4 in CVD are conflicted. Therefore, we aimed to evaluate the relationship between RBP4 with the presence and severity of coronary artery disease (CAD) in this study.55 patients with presenting acute coronary syndrome (ACS) and 43 control subjects who had various cardiovascular risk factors with normal coronary artery on coronary angiography were included in this study. The serum RBP4 concentrations were measured using ELISA method, clinically and anatomically score models were used to assess the severity of coronary lesion.Serum RBP4 levels were significantly higher in patients with ACS compared to the without ACS . RBP4 was correlated with GENSINI and SYNTAX I score . However, there was no relationship between RBP4 and GRACE score.The serum RBP4 levels increase in patients with CAD and its increased levels may be correlated with CAD severity. Coronary artery disease (CAD) has high mortality and morbidity rate in the world and one of the main mechanisms responsible for its pathophysiology is inflammation There are many studies showing high RBP4 levels in obesity, hypertension and insulin resistance closely related with CAD 2), severe liver disease were the exclusion criteria.This observational case-control study was carried out with 55 consecutive patients with ACS who admitted to the emergency department of Pamukkale University Faculty of Medicine Hospital, and 43 control subjects with normal coronary artery who underwent coronary angiography between January 2018 and June 2018. Inclusion criteria were the age range of 19-90 years, performing coronary angiography due to ACS or performing coronary angiography due to positive and suspected ischemia in non-invasive stress imaging tests in patients with at least one cardiovascular risk factor such as hypertension, diabetes or smoking. Previous myocardial infarction or CAD history, cardiomyopathies, severe heart valve diseases, acute pericarditis/myocarditis, cerebrovascular diseases, malignancies, hematological diseases, acute and chronic infections, chronic inflammation, autoimmune diseases, chronic renal failure  was diagnosed on the basis of typical symptoms of myocardial ischemia , newly developed ischemic ST-T changes (ST-elevation or ST-segment depression or prominent T-wave inversion) in at least 2 contiguous ECG leads and elevated cardiac-associated biomarkers of necrosis in an appropriate clinical presentation.This study was consistent with the Helsinki declaration and was approved by the hospital's ethical review board . Written informed consent form was obtained from all subjects.Peripheral venous blood samples were collected after 8-12 hours fasting from all subjects. Routine biochemical parameters including fasting blood glucose, kidney function tests, lipid parameters and C-reactive protein (CRP) were analyzed with Cobas 8000 Autoanalyser  and hematologic parameters were measured using Mindray BC-6800 Autoanalyser .The serum concentration of RBP4 was measured using a commercially available enzyme-linked immunosorbent assay kit  in accordance with the manufacturer's instructions. Samples were diluted 2-fold into the dilution buffer provided. The detection limit of the assay was 0.26 mg/L (range: 0.5-180 mg/L). All samples were assayed in duplicate. The absorbance was measured at 450 nm with Biotek Elx800 microplate reader . The data were processed with the Gen5 Data Analysis software  and RBP4 results were calculated based on the standard curve generated by using a five-parameter curve fitting equation and multiplied by the sample dilution factor.Transthoracic echocardiography was performed with Vivid 7 GE  in the left lateral decubitus position and left ventricular morphology and functions were evaluated. The left ventricular ejection fraction was calculated by using bi-planar Simpson method.All patients underwent transfemoral angiography using the standard Judkins technique. Diagnostic angiograms were recorded using a digital media viewer and the records were reexamined by 2 experienced cardiologists who were blind to the study protocol.www.syntaxscore.com) Global Registry of Acute Coronary Events (GRACE) score including 8 parameters  was used for the clinical risk assessment All data were analyzed using SPSS v.21.0 for Windows . Categorical variables were expressed as frequencies and percentages; continuous variables were expressed as means ± SD. The normal distribution of the data was evaluated using the Kolmogorov-Smirnov test. The variables with normal distribution were compared using Student T test and Mann-Whitney U test was used when the normal distribution was not provided. Comparison of categorical variables was performed with chi-square test. The correlation between variables was tested via Spearman or Pearson correlation analysis and p <0.05 was considered as statistically significant.The demographic-clinical characteristics and other findings of the study population are summarized in .The mean age and gender distribution differed among groups  vs. 40%, The average GRACE, GENSINI SYNTAX I scores were calculated as 115.62 ± 24.24, 66.41 ± 50.53 and 27.30 ± 9.73 respectively in patients with ACS. There was a positive correlation between RBP4 with GENSINI score  and SYNTAX I score (r = 0.403 and p = 0.002). However, it was not found any relationship between RBP4 and GRACE risk score .We evaluated the role of RBP4 in determining the presence and severity of CAD in this study. We found that the serum RBP4 levels were higher in ACS patients than in controls. Also GENSINI and SYNTAX I scores showed significantly correlation with RBP4, but not GRACE score.Atherosclerosis usually develops with chronic vascular inflammation and one of the inflammation source is cellular oxidative stress. It has been suggested that RBP4 may be associated with oxidative stress and Farjo et al. confirmed that RBP4 initiates the process by stimulating gene expression associated with endothelial inflammation Unlike our study, some studies have failed to show relationship between CAD with RBP4. In Korean adults who underwent coronary angiography due to stable angina pectoris, the RBP4 level did not differ between patients with and without CAD 2 calculated by Cockcroft-Gault formula. It is expected that increased levels of RBP4 may affect lipid parameters that pose a risk for CVD It has been shown that serum RBP4 levels increase with deterioration of renal function but we failed because of not including subjects with GFR<60 mL/min/1.73 mThe major limitation of our study was the cross-sectional design, which prevented us from inferring cause-effect relationship of RBP4 with CAD. Also we did not asses the long term clinical outcomes of increased serum RBP4 levels in ACS patients. This study had relatively small sample design and we could not rule out the effects of pharmaceutical agents on RBP4 levels because the majority of subjects with various cardiovascular risk factors were under treatment. The control group had high probability of CAD so we may not extrapolate our conclusions to healthy subjects. The groups were not matched for age and gender and this may affect serum plasma RBP 4 level. Finally, methodological differences in measuring serum RBP4 levels may change the outcomes of the studies.Serum increased RBP4 levels may show the presence and severity of CAD. However, the large multi-center studies are needed to better determine the role of RBP4 in CAD and to evaluate its prognostic significance.Funding: None.All the authors declare that they have no conflict of interest in this work.


Significant (up to 30% e.e.) chiral induction during nucleophilic fluorination to form β-fluoroamines using oxazaborolidine (pre)catalysts and CsF also can be achieved. Screening different boranes revealed a correlation between calculated fluoride affinity of the borane and nucleophilic fluorination reactivity, with sufficient fluoride affinity required for boranes to react with CsF and form Cs[fluoroborate] salts, but too high a fluoride affinity leading to fluoroborates that are poor at transferring fluoride to an electrophile. Fluoride affinity is only one component controlling reactivity in this context; effective fluorination also is dependent on the ligation of Cs+ which effects both the phase transfer of CsF and the magnitude of the [Cs⋯F-BR3] interaction and thus the B–F bond strength. Effective ligation of Cs+ (e.g. by [2.2.2]-cryptand) facilitates phase transfer of CsF by the borane but also weakens the Cs⋯F–B interaction which in turn strengthens the B–F bond – thus disfavouring fluoride transfer to an electrophile. Combined, these findings indicate that optimal borane mediated fluorination occurs using robust (to the fluorination conditions) boranes with fluoride affinity of ca. 105 kJ mol−1 (relative to Me3Si+) under conditions where a signficant Cs⋯F–B interaction persists.Despite the general high fluorophilicity of boron, organoboranes such as BEt Simple boranes with the optimal fluoride ion affinity are effective as catalysts for phase transfer nucleophilic fluorination with CsF. The established dogma is that boranes  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>Y multiple bond character; (ii) reducing the partial positive charge localised at boron using less electron withdrawing substituents, and (iii) increasing the pyramidalisation energy at boron.6 The first two points combined explains the trend in the fluoride affinity of the simple  boranes: BF3  ≫ trialkylboranes (FIA of BMe3 = 132 kJ mol−1) > B(OH)3 .6 Despite the facile ability to tune fluoride affinity at boron there are no reports, to the best of our knowledge, that utilise low FIA boranes as catalysts for MF phase transfer fluorination. Due to the importance of fluorinated molecules in pharmaceuticals and agrochemicals7 and the attractive nature of using metal fluoride (MF) salts and simple boranes to effect nucleophilic fluorination, we sought to: (i) demonstrate that low fluoride affinity boranes can be used as MF phase transfer catalysts and (ii) develop the structure activity relationships key to enabling this reactivity.To expand the electrophile scope amenable to fluorination with fluoroborates requires an understanding of the factors controlling the fluoride ion affinity (FIA) of boranes, thereby enabling its rational modulation. Analysis of calculated FIA values reveals that borane fluorophilicity can be attenuated by: (i) the presence of significant B8,9 Established phase transfer agents include metal chelators (e.g. cryptands), organic cations (e.g. [R4N]+),9 Lewis acids that weakly bind fluoride (e.g. in hypercoordinated silicates) and compounds that function as multiple hydrogen bond donors to fluoride, e.g. bis-ureas.8,9 Highly notable recent work using the latter class also achieved excellent (>85% e.e.) enantioselectivity during phase transfer nucleophilic fluorination of certain alkylhalides  with MF.8 Boranes with low FIA (relative to BF3) have been largely overlooked in this area. Even the stoichiometric use of fluoroborates derived from lower fluoride affinity boranes in nucleophilic fluorination is rare, with the very limited exceptions including: the use of PinBF in the ring opening fluorination of epoxides;10 the use of fluoroborate A (11 the use of Mes2B(aryl) compounds to bind, and on addition of [CN]−, to release fluoride.12 Note, when using compound A (or when adding an exogenous nucleophile to [Mes2B(aryl)F]−), the formation of a B←SR2 dative bond (or a B–CN bond) contributes to making fluoride transfer from boron to carbon thermodynamically favourable. This factor will be absent using Lewis base free conditions/boranes in MF phase transfer/nucleophilic fluorination cycles  necessitates their use.borate A , top to n cycles , bottom.13 in enantioselective nucleophilic fluorination.Herein we demonstrate that simple (and Lewis base free) boranes are useful CsF phase transfer fluorination catalysts. Furthermore, we have elucidated important factors controlling the effectiveness of low FIA boranes as CsF phase transfer fluorination catalysts. Demonstrating that simple boranes can act as CsF phase transfer fluorination catalysts opens the door to using the plethora of readily synthesised enantioenriched boranes+) carbon electrophiles, as suggested by previous computational studies.14 For these initial studies [NMe4]+ salts were used to minimise any complications associated with strong interactions between anion and cation. In contrast, significant R3B–F⋯M  interactions are expected, particularly in weakly coordinating solvents, which could modify fluorination reactivity using M[R3BF] salts. [NMe4][FBPh3] was synthesised by combination of BPh3 and [NMe4][F] and combined with [Ph3C][B(C6F5)4]. This resulted in fluoride transfer from boron to carbon as indicated by 11B (change in δ11B from 3.4 for [FBPh3]− to 60.5 for BPh3) and 19F NMR spectroscopy . The use of the ethyl congener, [NMe4][FBEt3], resulted in an analogous outcome (BEt3 and Ph3CF formation). Therefore in contrast to [BF4]− (which does not transfer fluoride to Ph3C+), these [R3BF]− anions do transfer fluoride to Ph3C+ .Initially we sought to determine if the fluoroborates derived from low fluoride affinity triorganoboranes will transfer fluoride to weaker .To guide subsequent studies and identify other boranes with potential as phase transfer fluorination catalysts we calculated fluoride ion affinity values using a closely related method to that reported by Greb 3 at this level provided for comparison. The calculations were consistent with the expected outcomes e.g. electron withdrawing groups (in 1-3) increase fluoride affinity (relative to PhBPin). While increased multiple bond character, e.g. BNR2 double bond character being greater than BOR double bond character, leads to CBS (Corey–Bakshi–Shibata) oxazaborolidine catalyst 4 being a weaker Lewis acid towards fluoride than PhBPin. Several boranes with very similar calculated fluoride affinity values also were identified to probe the effect different functional groups (e.g. NO2vs. CF3 in 1 and 3x), ortho vs. meta vs. para substitution (in 3x) and substituent size (e.g. BEt3vs.5) have on borane reactivity towards MF and the subsequent reactivity of the fluoroborate. This is important as in contrast to [R4N]+, solvation of M+ and F− needs to be considered along with the effect of strong interactions between M+ and the fluoride of the fluoroborate persisting in solution.These calculations  enabled 3 as catalyst) led to no fluorination in CHCl3, thus all further fluorination studies were performed using CsF. The disparity between KF and CsF is attributed to the greater lattice energy of KF relative to CsF effecting the energetics of the reaction with borane (vide infra). It is noteworthy that the use of ground CsF led to substantial rate enhancements versus reactions using as received CsF. This is consistent with an increase in surface area facilitating the phase transfer reaction between solid CsF and the dissolved borane. Ground and dried CsF is used throughout this study. With both BEt3 and ArBPin based boranes haloalkane solvents gave better outcomes than other solvents, e.g. MeCN, thus only results in DCM or chloroform are discussed in depth. Anhydrous conditions are essential, as the presence of water  led to a significant retardation in the rate of fluorination of 6 using 1. The use of protic additives was not explored with BR3 species due to their propensity to undergo protodeboronation with ROH. Finally, a control in the absence of borane led to no fluorination of 6 with CsF in chloroform.Fluorination of 6 to form β-fluoroamine, 7, using MF (M = K or Cs) catalysed by boranes was explored as a test reaction to determine if there is any correlation between borane fluoride affinity and phase transfer/nucleophilic fluorination reactivity . Attempt3 and 1. This demonstrates that borane phase transfer catalysts can be used to access important fluorinated molecules.8 As expected the identity of the borane is all important, with weaker Lewis acids e.g. PhBPin, and stronger Lewis acids (e.g. BPh3) both giving poorer outcomes. The former is consistent with a minimum fluoride affinity being required to form the Cs[fluoroborate] salt, while the latter indicates that if the fluoride affinity is too high then this disfavours transfer of fluoride from boron in the fluoroborate to the electrophile (fluoroborate formation is observed with the higher FIA boranes). However, there are additional factors beyond fluoride affinity controlling fluorination using boranes, as 3p was a relatively poor catalyst despite having an identical calculated fluoride affinity to 1. Furthermore, the meta and ortho derivatives, 3m and 3o were more active than 3p, despite similar FIA values. Finally, a Hammett analysis  boranes led to effectively no correlation, indicating other effects are impacting the fluorination outcome (vide infra).From this borane scoping, phase transfer fluorination of 6 using CsF was most effective with 10 mol% BEt3 and 1 as catalysts and this revealed the fluoroborates derived from these boranes to be poorer sources of fluoride relative to the Lewis base incorporated borate A. For example, no fluorination of octyl bromide or benzyl halides was observed even after prolonged periods refluxing with excess borane/CsF  did undergo fluorination with CsF using 1 or BEt3 as catalysts. Reaction of β-bromo sulphide 8 with CsF with either BEt3 or 1 as catalyst in CHCl3 led to significant formation of stilbene (mixture of cis–trans isomers) with only traces of 9 formed. Serendipitously, we found that the outcome of this reaction is effected dramatically by solvent. Using DCM/n-hexane (6 : 1) as the reaction medium, stilbene formation was negligible (ca. 3%) and 9 could be formed in moderate yield using BEt3  fluoride source [NMe4]F (in the absence of any borane) under identical conditions led to significant stilbene formation (2 : 1 ratio of stilbene : 9) in contrast to the outcome using CsF/BEt3. The reaction of Ph3CCl with CsF in CHCl3 catalysed by either BEt3 or 1 proceeded in moderate to good yield. Benzoyl chloride proved to be more challenging, with 1 as the catalyst fluorination proceeded to only ca. 5% conversion. However, using 10 mol% BEt3 benzoyl fluoride was formed in good yield.Stronger electrophiles .8 While 5 was ineffective as a catalyst in halocarbon solvents, it did function in the presence of MeCN. However, the use of stoichiometric Cs[5-F] in DCM/MeCN mixtures while leading to formation of 7 and 9, resulted in no e.e. being observed by chiral HPLC analysis. Furthermore, significant amounts of hydrodehalogenation also was observed using Cs[5-F] alongside formation of 7/9, possibly via a mechanism related to the Midland reduction .15 In addition to 7, ca. 5% of the β-amino-alcohol, 10 , including resonances consistent with products derived from reaction of 4/11 with water as previously reported  also was explored as it is not prone to loss of hydride. Surprisingly , as received 4 effectively catalysed fluorination of 6 with CsF and led to appreciable e.e. in 7  than the commercial material was achieved.18 These higher purity CBS catalysts gave worse outcomes than using commercial batches of 4 in the fluorination of 6 with CsF. In addition, all >90% purity CBS catalysts  displayed an induction period before significant fluorination occurred .While this work with CBS (pre)catalysts provides proof of principle that enantioselective borane phase transfer fluorination catalysis is feasible, the ill-defined and complex mixtures produced using CBS (pre)catalysts under these conditions is a complicating factor presumably contributing to the maximum e.e. being 30%, despite using multiple CBS (pre)catalyst structures. This highlights the importance of using borane catalysts that are robust under these conditions to allow for rational control of reactivity  was observed on addition to KF suspended in CHCl3, consistent with the higher lattice enthalpy of KF relative to CsF .19 The absence of any fluoroborate formation is presumably why there is no fluorination of 6 using these boranes and KF. In contrast, combining BEt3 with CsF formed the fluoroborate in a range of solvents (3] were significantly different in DCM/CDCl3 (entries 1 and 2) compared to those in MeCN (entry 3), with this solvent dependence attributed to a different aggregation of the Cs[FBEt3] salt. This is supported by DOSY NMR studies which indicated [FBEt3]− was a monomer in MeCN, but exists in larger aggregates in DCM ([Cs(FBEt3)]n with n >1, vide infra). This is attributed to MeCN being more effective at ligating Cs+ than halocarbon solvents, breaking up Csn(μ-F)n (n >1) units. A related process would explain the addition of [2.2.2]-cryptand (1.25 eq.) to Cs[FBEt3] in halocarbon solvents resulting in a considerable shift in fluoroborate resonances (compare entries 2 and 4). The cryptand by strongly binding Cs+ will weaken the B–F⋯Cs interaction which will increase the B–F bond strength (vide infra).To understand why only certain borane/MF combinations are effective fluorination catalysts, their ability to form M[fluoroborate] salts was explored. With BEtsolvents . Notably+ than MeCN (confirmed by addition of [2.2.2]-cryptand to a MeCN solution of Cs[FBEt3] causing a shift from δ19F = −178.9 to δ19F = −190.2 (entry 3 vs. 5) indicating displacement of MeCN from Cs+ by cryptand). The different chemical shifts and coupling constants observed suggests significantly different B–F bond strengths in these systems, presumably due to different Cs⋯F–B interactions. Therefore Cs+ ligation will effect not just the energetics of solid to solution phase transfer of CsF using boranes, but also the ability of the formed Cs[FBR3] to act as a nucleophilic source of fluoride. The NMR data indicate that CsF/BR3 in halocarbon solvents (e.g. entries 1/2) should be the most nucleophilic source of fluoride using BEt3 as catalyst, due to the downfield shifted 11B resonance . This is consistent with the catalytic fluorination results where halocarbon solvents gave better outcomes than using MeCN.As expected, [2.2.2]-cryptand more strongly ligates Cs3 but a different environment around the boron centre, which significantly impacts its performance in catalysing nucleophilic fluorination (vide supra). Compound 5 showed no propensity to bind CsF in halocarbon solvents (by NMR spectroscopy) in contrast to BEt3, consistent with the disparate catalytic nucleophilic fluorination performance observed in DCM. This further confirms that calculated fluoride affinity values must be used with caution for predicting reactivity when there is a coordinating cation present. Using DCM/MeCN mixtures or neat MeCN did enable formation of the fluoroborate, Cs[5-F] 2} units propagated into a 1D-coordination polymer by three acetonitrile molecules bridging two adjacent caesium centres Cs–F-Baryl3  Å and Cs⋯F = 3.034 Å, whereas in Cs[5-F]: B–F = 1.524(5) Å and Cs⋯F = 2.945(3) Å. This is consistent with: (i) the presence of a more Lewis acidic caesium centre more strongly interacting with the B–F unit, thereby reducing the B–F bond strength; (ii) the observed impact of caesium ligation (e.g. with cryptands – vide infra) on the ability of fluoroborates to transfer fluoride from boron to carbon electrophiles. The low formal coordination number of Cs+ in Cs[5-F] may explain the disparity in reactivity between 5 and BEt3 towards CsF, particularly in halocarbon solvents. The larger hydrocarbyl groups in 5 (relative to Et in BEt3) may prevent additional interactions to Cs+  thus leading to unfavourable solvation energetics (and thus no reaction) when 5 is combined with CsF in halocarbon solvents. This again emphasises that appropriate ligation of caesium in Cs[F–BR3] is vital alongside the appropriate borane fluoride affinity in enabling borane catalysed phase transfer fluorinations.Single crystals of Cs[5-F] were obtained from a saturated MeCN solution at −25 °C with its solid state structure consisting of {Csaryl3 B; , in whicversus an internal standard which showed no decrease in the amount of PhBPin present in halocarbon solutions). The disparity can be attributed to the lower fluoride affinity of PhBPin which will disfavour reaction with CsF and is presumably why PhBPin is a poor catalyst for nucleophilic fluorination of 6.Moving to dioxaborolanes, with ArBPin/CsF combinations only the free ArBPin was visible by NMR spectroscopy in halocarbon solvents, although solid is present in these reactions. Assessing these mixtures by NMR spectroscopy using an internal standard revealed a significant decrease in the intensity of ArBPin resonances on addition of CsF for 1 (and 2). This indicates the formation of poorly soluble  fluoroborate salts derived from 1 (and 2). Thus 1 does react with CsF consistent with its ability to catalyse fluorination. In contrast, no evidence for formation of the fluoroborate was observed on combining CsF/PhBPin , despite 3p having an effectively identical calculated fluoride affinity to that for 1. This is consistent with the relatively poor catalytic performance of 3p in the fluorination of 6  interaction with the Cs+ cation in the fluoroborates derived from 1 and 3p. This is attributed to intramolecular ArCF3⋯Cs+ interactions using meta substituted 1 persisting in solution, in contrast intramolecular ArNO2⋯Cs+ contacts are not feasible in para substituted [3p-F]Cs (as Cs⋯F–B contacts are expected to be preferred based on the structures of B and C). Multiple short ArCF3⋯Cs contacts are present in the solid-state structure of the closely related salt Cs[FB(neop)(m-C6H3(CF3)2)] . A similar effect also was observed when comparing the ortho and para isomers of ((CF3)C6H4)BPin, 15o and 15p. Borane 15o was significantly more active as a catalyst in the fluorination of 6 with CsF (conditions as per ca. 10% 7 formed after 8 h). This is consistent with 15o forming ca. 15% [15o-F]Cs in chloroform whereas 15p displayed no propensity to bind CsF under identical conditions. Note, 15o and 15p have effectively identical calculated FIA values  again indicating that the ability of borane substituents to ligate Cs+ plays an important role facilitating CsF phase transfer. While intramolecular ligation of Cs+ in the Cs[fluoroborate] salt is clearly beneficial for enhancing the phase transfer of CsF by boranes with ortho/meta CF3 and NO2 groups, stronger Lewis basic ortho substituents actually lead to poorer outcomes. For example, using ((o-NH2)C6H4)BPin led to much slower fluorination of 6 (68% 7 formed after 48 h).The importance of intramolecular ligation of Csrination . This waectively , right. ectively . Again tδ11B = 2.9 and δ19F = −144.4, albeit both resonances being broad with no resolved B–F coupling. The upfield shift (relative to entry 8) in δ11B suggests adding cryptand leads to stronger B–F binding, presumably by weakening the Cs⋯F–B interaction. This should disfavour nucleophilic fluorination by the fluoroborate, which indeed is what was observed. Specifically, the use of a 1 : 1 combination of 1/[2.2.2]-cryptand retarded fluorination of 6 with CsF  but also to maintain a significant Cs⋯F–B interaction that labilises the B–F bond (favoured by weaker binding of Cs+).To probe the consequences of caesium ligation in the BPin systems further, the effect of [2.2.2]-cryptand on Cs[fluoroborate] reactivity was explored. A mixture of 1/[2.2.2]-cryptand and excess CsF gave a halocarbon soluble product , entry 9+ to enable solid to solution phase transfer. However, avoiding too effective a ligation of Cs+ is also vital, as good ligation of Cs+ weakens the Cs⋯F–B interaction, strengthening the B–F bond and thereby leading to less reactive fluoroborates. In terms of predictability, boranes with calculated fluoride affinity of 95–120 kJ mol−1 (vs. Me3Si+) appear to be suitable candidates as nucleophilic fluorination catalysts, with the caveat that other factors (e.g. borane stability under the reaction conditions/forming the correct fluoroborate aggregation/Cs+ ligation level in solution) are also important to consider. Finally, weak intramolecular ligation of Cs+ by borane substituents appears an effective method to enable lower FIA boranes to achieve CsF phase transfer and nucleophilic fluorination. When the various prerequisites are met, simple boranes are effective catalysts for nucleophilic fluorination using CsF, including to access useful products (e.g. β-fluoroamines).Despite the high fluorophilicity of boron, certain organoboranes and boronate esters can be employed as CsF phase-transfer nucleophilic fluorination catalysts. Chiral induction during fluorination with borane catalysts also was demonstrated as proof of principle (up to 30% e.e.), however limited catalyst stability under these reaction conditions precluded realising high e.e. with CBS systems, highlighting the importance of using boranes robust to fluorination conditions. Regarding the factors controlling effective catalysis, as expected, nucleophilic fluorination reactivity is impacted by B–F bond strength, which is dependent on borane Lewis acidity towards fluoride. Sufficient fluoride affinity favours the borane reacting with CsF, however if fluoride affinity is too high the resultant fluoroborate does not effectively transfer fluoride to electrophiles. Importantly, nucleophilic fluorination is most effective under conditions that provide sufficient ligation of CsFull experimental procedures, NMR spectra, DFT and crystallographic details/data are provided in the ESI.†3 and 5 catalysed fluorinations. KY performed all the computational investigations. MU collected and solved the crystal structure. SK and MI drafted, reviewed and edited the manuscript.MI and SK conceived the research concept and aims and analysed all data. SK performed the majority of the synthetic work and the majority of the analytical components of this project. MP performed preliminary investigations on commercial CBS, BEtThere are no conflicts to declare.SC-013-D2SC00303A-s001SC-013-D2SC00303A-s002


We examined how presentations of organ donation cases in the media may affect people’s decisions about organ donation issues. Specifically, we focused on the combined effect of the information about the number of recipients saved by the organs of one deceased person (one vs. four) and the identifiability of the donor and the recipient(s) in organ donation descriptions, on people’s willingness to donate the organs of a deceased relative. Results suggest that reading about more people who were saved by the organs of a deceased donor does not increase willingness to donate. Replicating earlier research, we found that reading about a case of organ donation involving an identified deceased donor, deceased willingness to donate. However, this effect was attenuated when participants read about more recipients who were saved by the donation. Importantly, the presentation that prompted the greatest willingness to donate a deceased relative’s organs was the one that featured an unidentified donor and only one identified recipient. Finally, an explorative investigation into participants’ subconscious thoughts of death following the organ donation story revealed that identifying a deceased organ donor prompts more thoughts of death in the perceiver (regardless of the number of recipients). Moreover, we often encounter—in the printed press, online, or in television reports—of cases of organ donations with information about a deceased donor and about one or several recipients whose lives were saved by that donation.How might these ads and stories affect readers? In a previous study , we demoscope neglect occurs in this context), and to learn about the underlying mechanism . From the practical standpoint, it will help in identifying the best way to present the issue of organ donations in the media, in a manner that encourages people’s willingness to donate organs.In the present research, we take one step further in investigating the impact of the presentation of organ donation cases in the media on people’s WTD organs, by examining the role played by the number of recipients saved by the organs of one deceased person, and whether learning about more recipients who were saved as a result reduces thoughts of death, thereby increasing support for organ donation. In addition, we sought to examine the combined effect of the number of recipients saved by the organs of one deceased person and the identifiability of the donor and the recipient[s] in people’s decisions about organ donation issues. Answering these two questions has the potential to make both a theoretical and a practical contribution. First, this investigation will help in understanding the role played by the number of people saved by organ donation, in organ donation decisions . Thus, when a specific case of a prospective organ donation recipient is presented, it can only be by way of illustration, rather than as an actual request for help .Moreover, when people consider the issue of organ donations, they are confronted with the disturbing thought of their own demise, or that of a relative. According to terror management theory e.g., , prosociidentifiability effect in the context of organ-donation decisions. However, in that study, the recipient was always a single individual, and the donated organ was always a kidney. The research on scope insensitivity and on the singularity effect of identified victims, as reviewed above, raises the question of whether presenting more than one individual who has been saved by organ donations would boost support for organ donations among the public.To the best of our knowledge, This question is important from a theoretical perspective, since while stories about several individuals being saved by the donation of organs of a deceased person may boost organ donations—by prompting thoughts about the lives being saved (rather than about death) —they mayIn light of recent appeals for organ donations that highlight the fact that one dead person can save the lives of nine people, it is also important to examine this strategy from a practical perspective.In the present research, we sought to examine the combined impact of the identifiability of the donor and the recipient, and their number (one vs. four recipients) on organ-donation decisions. To this end, we chose to focus on the decision to donate the organs of a deceased close relative (rather than one’s own), since it covers all prospective donors, including those who are willing or have already committed to donate their own organs after death.In light of the findings of When the deceased donor is left unidentified, we expected identifying information about only one prospective recipient to prompt greater support for organ donations, especially when only one such recipient is presented—in line with the research on the singularity effect, which states that people are more likely to sympathize with, and tend to take the perspective of, a single identified victim, than when a group of such victims with the same need are involved.To examine these predictions, we used the study design used by G*Power computer application (f = 0.15), with a power of 80%. Accordingly, we recruited 304 undergraduate students at Ben-Gurion University , through an online subject pool in exchange for monetary prizes—to complete a short survey online. Participants were randomly assigned to one of eight experimental conditions, in a 2 × 2 × 2 design of Donor’s Identification (identified vs. unidentified), Recipient’s Identification (identified vs. unidentified), and the Number of Recipients (1 vs. 4), as explained below.To determine the number of participants to recruit for the study, we conducted a power analysis by means of the lication . This inYes/No).Participants first read a story (adopted from 1 (Strongly disagree) to 7 (Definitely agree).Next, participants were asked to imagine that a close relative of theirs had just died, and that the hospital’s medical staff were asking their family to consider donating his organs to save the life of someone waiting for transplantation. Participants were then asked to rate their WTD their deceased relative’s organs on a seven-category scale ranging from Yes/No).Finally, they were asked to provide demographic information about themselves, including ratings of their degree of religiosity, a variable found in previous studies to be related to willingness to donate organs  and to indicate whether they themselves were registered organ donors (p = 0.80), nor under the different photos used to identify the donor and the recipient (p = 0.85). We therefore analyzed the Single Recipient condition beyond the different organs and photos.Willingness to donate the relative’s organs did not significantly differ under the different organ conditions used in the Single Recipient condition  was found to play a significant role in the decision about donating the organs of a deceased relative, and may interact with the different presentations , we usedM = 6.29, SD = 1.05) than unregistered people , F = 135.93, p < 0.001, ηp2 = 0.31. No other significant main effects were found.Results revealed a significant main effect for consent status—such that, as expected, registered donors expressed greater WTD the organs of a deceased relative  = 5.77, p = 0.017, ηp2 = 0.02. As illustrated in M = 5.53, SD = 1.67) were less willing to donate the organs of a deceased relative than those whose account talked about an unidentified donor ; F = 6.09, p = 0.014, ηp2 = 0.02. However, when four recipients were saved by the organs of the one deceased donor, identifiability of the donor had no significant effect on willingness to donate—F = 0.85, p = 0.36, ηp2 = 0.003. This suggests that knowing about several people who were saved by the organs of a single dead donor attenuates the effect of Donor’s Identification in reducing support for organ donations. However, reading about four people who were saved by the organ donation did not have a significant effect in increasing WTD.The interaction between Donor’s Identification and Number of Recipients was significant F = 5.99, p = 0.015, ηp2 = 0.02. As evident in F = 5.93, p = 0.015, ηp2 = 0.02; while in the Unidentified condition no significant difference was found between one recipient and four recipients, F = 0.96, p = 0.33, ηp2 = 0.003. This result is in line with previous research on the singularity effect in charitable giving, which suggests that a single identified recipient prompts a greater WTD than a group of recipients. Another way to look at the interaction is to examine the effect of identifiability of a single target and that of a group of four on WTD. A simple effect analysis reveals that identifying four recipients, actually decreased WTD , compared to four unidentified recipients , F = 7.23, p = 0.008, ηp2 = 0.024. However, the role of the recipient’s identifiability was far from significance when only one recipient was presented  = 0.47, p = 0.49, ηp2 = 0.002). This finding is interesting, since it highlights the notion that identifiability may have a negative effect on WTD when several targets are presented (rather than only one). It is possible that providing too much information about several people and various transplanted organs increases stress among the perceivers, distancing them from the situation  = 3.23, p = 0.069, ηp2 = 0.011. As illustrated in M = 5.54, SD = 1.67) than when the donor is unidentified ; F = 4.17, p = 0.043, ηp2 = 0.024. When four recipients are presented, no significant effects were found, and the main effect of Recipient’s Identification approached significance, suggesting that four unidentified recipients encouraged greater WTD  than four identified ones  F = 2.75, p = 0.099, ηp2 = 0.02. Previous research on the role of the identifiability of a group of recipients in promoting monetary donations found mixed results: in some studies, it had no effect on donations, while in others it decreased them  = 2.15, p = 0.14).Finally, the three-way interaction between donor’s identifiability, recipients’ identifiability and the number of recipients approached significance sed them ,b. RepliF = 2.46, p = 0.018, ηp2 = 0.055. Post hoc analysis suggests that participants who were told about an unidentified donor and one identified recipient were significantly more WTD than participants in most of the other conditions, as reported in Judging by One key finding of Study 1 is that being told about four recipients who were saved by the organs of a single deceased donor attenuates the effect of Donor’s Identification in reducing the willingness to donate. Since previous research  suggestsfour recipients were saved by the organs of the deceased, this effect was attenuated, such that their WTD was not significantly different from that of participants who had been told about an unidentified donor.Study 2 was an exploratory attempt to examine the psychological mechanism that may explain the interaction between identification of the donor and the number of recipients, in terms of the participants’ WTD, as found in Study 1. As noted, previous research suggests that the identifiability of the donor prompts thoughts of death, rather than about saving lives, resulting in diminished WTD. In Study 1, we found that donor identifiability reduced WTD when only one recipient was saved by the organ donation—but when participants were told that In Study 2, we examined the salience of death-related thoughts in people’s minds after reading about a case of organ donation. As in Study 1, participants were given a written account about a recent case of a young man who had been killed in a car accident, whose organs were donated to save the lives of others. The study included a 2 × 2 design, manipulating the Donor’s Identification (identified vs. unidentified) and the Number of Recipients (one vs. four) in the story. However, in this case, to keep the design simple, the recipients in all conditions were unidentified. We then examined the participants’ subconscious thoughts of death after the various descriptions, by means of a word-completion task.death; funeral; grave; body; deceased; mourning; and “Shivah” (a week-long mourning period in Judaism). The dependent measure was the number of death-related words with which a participant completed the fragments. Finally, participants provided their demographics including information about whether they are registered donors (yes/no), and religiosity rating (as in Study 1).Four hundred and forty undergraduate students at Ben-Gurion University (from a computerized pool of subjects) took part in the study: 63% females, Mage = 26.56, SD = 13.32. Participants were randomly assigned to one of four groups in a 2 × 2 design manipulating the identifiability of the donor (identified vs. unidentified) and the number of organ recipients (one versus four). As in Study 1, participants first read about a young man who had been killed in a car accident, with or without identifying information. They next read that the organ[s] of this man saved the lives of one [four] young men who urgently required them. To examine participants’ subconscious death thoughts, we used a word-completion task involving words that could be completed with either neutral or death-related words. This procedure has been used successfully in previous research to examine people’s accessibility of various subconscious contents e.g., . The worIn this study we examined accessibility to death-related words after reading about the case of organ donation without assessing WTD, building upon the relationship between thoughts of death and WTD after reading about an identified versus unidentified donor found in previous research , since sM = 1.37, SD = 1.07. A two-way ANOVA on the number of death-related words by the two independent variables (identifiability and number of recipients) was conducted. Results reveal a significant main effect for Donor’s Identification—F = 4.17, p = 0.04, ηp2 = 0.01—such that reading about an identified deceased donor prompted more thoughts of death among participants  than reading the same story with an unidentified donor . The Number of Recipients fell far short significance F = 0.40, p = 0.85, ηp2 < 0.001. Although the interaction between identifiability and the Number of Recipients was not significant F = 0.82, p = 0.37, ηp2 = 0.002, in light of the results of Study 1, we looked at the effect of Donor’s Identification in each of the Recipient Number conditions separately. Simple-effect analysis revealed that Donor’s Identification increased thoughts of death in the One Recipient condition only F = 4.32, p = 0.038, ηp2 = 0.01, while in the Four Recipients condition Donor’s Identification had no significant impact on thoughts of death, F = 0.65, p = 0.42, ηp2 = 0.001. Holding Consent Status and level of religiosity constant in the analysis revealed similar results. Specifically, the main effect of Donor’s Identification remained significant (p = 0.049) while Consent Status (p = 0.56) and Religiosity (p = 0.31) did not reveal significant results.The number of death-related words completed by the participants in condition is presented in The results of our investigation of the effect of the presentation of organ donation cases on people’s WTD the organs of a deceased relative, replicated those of previous research by showing that when the participants read about a case of organ donation involving an identified deceased donor, their WTD diminished. However, it also yielded innovative findings about the effect of the number of recipients saved by a single deceased donor on people’s WTD the organs of a deceased relative. As with monetary donation decisions e.g., , we founOur explorative investigation into participants’ subconscious thoughts of death following the organ donation story replicated previous findings that identifying a deceased organ donor prompts more thoughts of death in the perceiver . While pIn the present research, thoughts of death and WTD were not examined in the same study, since several prior pilot studies  showed that only the task that is closely linked to the story (and to the identification manipulation) was influenced by the manipulation—subsequent tasks were not. Future study is therefore needed to further examine the possible role played by thoughts of death in mediating the link between Donor’s Identification and support for organ donation, perhaps by using physiological measures.Our research has a number of limitations that should be considered when drawing conclusions from it, or when planning related research. First, the experiments were not pre-registered. Specifically, Study 2 was of an explorative nature, and included pilot studies to explore the effect of the order of the two tasks  on the participants’ responses. Thus, future research is needed to replicate these findings, and to examine the mechanisms underpinning the pattern we observed, by means of other methods of gaging thoughts about death. Second, the participants in our experiments are from relatively individualistic societies and cultures. Since the identifiable victim effect has been found mainly in Western cultures , future The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ben-Gurion University of the Negev. The patients/participants provided their written informed consent to participate in this study.IH and TK designed the research, and analyzed the data and wrote the manuscript. IH performed the research. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.