WWTR1(TAZ)–CAMTA1 is the only genetic alteration in approximately half of EHE, EHE is a genetically clean and homogenous system for understanding how the dysregulation of TAZ promotes tumorigenesis. Therefore, by using EHE as a model system, we hope to elucidate the essential biological pathways mediated by TAZ and identify mechanisms to target them. The findings of EHE research can be applied to other cancers that are addicted to high YAP/TAZ activity.Epithelioid hemangioendothelioma (EHE) is a rare vascular cancer that involves a gain-of-function gene fusion involving TAZ, a transcriptional coactivator, and one of two end effectors of the Hippo pathway. Although the activity of TAZ and/or YAP, a paralog of TAZ, is consistently altered in many cancers, genetic alterations involving YAP/TAZ are rare, and the precise mechanisms by which YAP/TAZ are activated are not well understood in most cancers. Because WWTR1(TAZ)–CAMTA1 gene fusion in at least 90% of cases. Recent investigations have elucidated the mechanisms by which YAP/TAP-fusion oncoproteins function and drive tumorigenesis. This review presents a critical evaluation of this recent work, with a particular focus on how the oncoproteins alter the normal activity of TAZ and YAP, and, concurrently, we generate a framework for how we can target the gene fusions in patients. Since EHE represents a paradigm of YAP/TAZ dysregulation in cancer, targeted therapies for EHE may also be effective against other YAP/TAZ-dependent cancers.The activities of YAP and TAZ, the end effectors of the Hippo pathway, are consistently altered in cancer, and this dysregulation drives aggressive tumor phenotypes. While the actions of these two proteins aid in tumorigenesis in the majority of cancers, the dysregulation of these proteins is rarely sufficient for initial tumor development. Herein, we present a unique TAZ-driven cancer, epithelioid hemangioendothelioma (EHE), which harbors a  The dysregulation of mitogenic and/or growth-suppressor signaling cascades is a hallmark of oncogenesis . One sucTwo serine–threonine kinases and two adaptor proteins constitute the core components of this pathway ,6,7,8,9.In comparison with the “Hippo-On” state, in a “Hippo-Off” state, inactive Hippo-pathway kinases yield unphosphorylated YAP/TAZ, which are active and therefore able to translocate to the nucleus. As neither YAP nor TAZ contain DNA-binding domains, these transcriptional coactivators require TEA domain 1–4 (TEAD1-4) transcription factors ,36,37,38Paradoxically, although the activity of YAP/TAZ in cancer is most commonly pro-tumorigenic, in certain cancers, YAP/TAZ activity can be antitumorigenic ,42,43. FEpithelioid hemangioendothelioma (EHE) is a rare vascular sarcoma that was originally described by Weiss and Enzinger in 1982 as a soft-tissue tumor with both a vascular morphology and a borderline histologic phenotype, with both benign and malignant features . ClinicaAttempts have been made to stratify EHE into either aggressive or indolent diseases ,48,50. IHistologically, EHE is characterized by the presence of cords or nests of epithelioid cells, which are cells that contain a rounded cell body ,50,52. TFurther highlighting its endothelial lineage, EHE expresses the endothelial-cell markers PECAM1, CD34, and ERG ,53. For WWTR1(TAZ)–CAMTA1  gene fusion .,57.WWTR1WWTR1(TAZ)–CAMTA1 fusion characteristic of EHE, a YAP1–TFE3 fusion was identified in a related lesion that we now refer to as YAP1–TFE3-rearranged hemangioendothelioma . Re. ReWWTR1survival ,50. Sinc1 fusion ,63,64.WWTR1–CAMTA1 fusion is present in nearly all clinical samples of EHE, much rarer fusions are still being identified. A case series of six samples of EHE that did not contain the WWTR1–CAMTA1 fusion identified two further alterations involving WWTR1: the WWTR1–MAML2 and WWTR1–ACTL6A fusions [WWTR1 fusion, but no 3′ partner was identified. Overall, this cohort of variant WWTR1-fused EHE had a large predilection for development in the heart muscle, which, altogether, is a rare location for the development of EHE. It is uncertain if these alternative fusions alter the clinical course of patients with EHE, and with the continued use of NGS in the diagnosis of EHE, it is likely that additional rare fusions will be identified in EHE.While the  fusions . FurtherWWTR1–CAMTA1 fusion. They further found that 57% of the EHE samples contained secondary mutations and, most commonly, alterations in CDKN2A/CDKN2B. Furthermore, in their cohort, patients with secondary mutations were more likely to have later-stage disease (stages III and IV). While this work has broadened our understanding of the mutational landscape of EHE, it is uncertain whether these secondary mutations mechanistically enhance tumorigenesis in a substantial way or whether their presence is solely a marker of late-stage disease. Nevertheless, this investigation has enhanced our view of the genetic alterations within this disease, and further clinical and mechanistic research is necessary to identify whether these mutations are associated with survival differences or altered treatment responsiveness.While there has been significant success in the identification of driver fusions within EHE, limited efforts have been made to identify secondary mutations, which may alter the disease phenotype. Seligson et al. specifically aimed to identify these secondary mutations and correlate them with clinical characteristics . ConcurrWWTR1–CAMTA1 gene fusion in EHE, we aimed to identify the mechanisms that underly tumorigenesis. WWTR1, which codes for the TAZ protein, is a transcriptional coactivator and end effector of the Hippo pathway [Following our discovery of the  pathway ,35. In c pathway ,68. CAMT pathway ,72,73,74 pathway . In contWhile there is a significant overlap between the biology of this YT fusion and the TC and YAP fusions that are seen in other cancers, two recent studies have shown that YT displays unique features that distinguish it from other YAP/TAZ fusions.YAP1 to the fourth exon of TFE3, or the first exon of YAP1 to the sixth exon of TFE3. However, this N-terminal portion of YAP still retains the TEAD-binding motif, and, thus, YT fusions can associate with the TEAD family of transcription factors , YAP1–MAMLD1 , YAP1–FAM118B , and YAP1–SS18  [Two recent studies have broadened our understanding of the mechanisms driving YT-mediated tumorigenesis. Szulzewsky et al. aimed to identify the mechanisms that drive tumorigenesis in multiple YAP fusions, including rcinoma) . In thisrcinoma) .Interestingly, when compared with other YAP fusions, there were multiple unique features of YT that differentiate it from other YAP fusions, or a hyperfunctional form of YAP. While YT significantly upregulated YAP/TEAD transcriptional genes, RCAS tumors driven by YT formed a unique transcriptional cluster compared with tumors driven by other YAP fusions. They further demonstrated that, while all YAP fusions and a hyperfunctional form of YAP (YAP S127A/S397A) bind to TEAD1-, TEAD2-, TEAD4-, BATF-, and RUNX-specific genomic sites, YT binds to a unique set of consensus sequences. Specifically, YT binds to the TFE3, MITF, and SP1 genomic binding sites, which are normally occupied by the wild-type TFE3 protein . MechaniIn a study published the following year, Merritt et al. (2021) aimed to identify the mechanism of action of YT fusion . This stOverall, these two studies have elucidated the significant role played by the TFE3 portion of the fusion protein, as it alters the DNA-binding profile, transcriptional landscape, and phenotype of the resultant tumors. Therefore, considering this fusion to be an overactive form of YAP is an oversimplification .While the functions of the N-terminal TAZ and YAP portions present in YAP/TAZ fusions have been extensively researched, only recently has our understanding of the functional contribution of the C-terminal portion of these fusions expanded. Merritt et al. aimed to elucidate how the C-terminuses (CAMTA1 in TAZ–CAMTA1 and TFE3 in YAP–TFE3) contribute to the tumorigenicity of the fusion proteins . InitialYEATS2 subunit being highly amplified in clinical non-small cell lung cancer samples [They further identified that these fusion proteins interact with a unique set of chromatin-remodeling proteins that are not utilized by TAZ or YAP. These include members of the SWI/SNF-, NUA4/TIP60-, and COMPASS-like complexes, as well as other monomeric transcriptional regulators and histone modifiers. One of the most intriguing clusters of interactors is the Ada-Two-A-containing complex (ATAC) chromatin remodeler. Specifically, ATAC is a multimeric protein complex that contains one of two histone acetyltransferases: KAT2A, or its paralog, KAT2B, as well as an additional KAT14 acetyltransferase subunit . While K samples . In MerrWwtr1 locus is converted into a Wwtr1–Camta1 locus through the utilization of a flip-excision cassette and Cre-recombinase [Wwtr1–Camta1 transcript remains under the control of the Wwtr1 promoter element, the epigenetic control mechanisms of the transgene are maintained. Tumors were observed in mice when TC fusion was induced using either a ubiquitously expressed Cre, Rosa26-CreERT2, or an endothelial-specific Cre, the Cdh5–CreERT2 driver. These tumors most commonly developed along the surface of the diaphragm and involved retroperitoneal structures, such as the kidney and pancreas. Lung, liver, and soft-tissue tumors were similarly identified, but at a lower frequency. These tumors were histological mimics of human EHE and expressed key EHE and endothelial markers, which were verified using IHC. To validate these tumors as replicas of the human EHE, we performed a whole-transcriptomic analysis of human EHE, as well as differential-expression analyses of other endothelial cell tumors and control tissues, and identified a gene set of 93 transcripts that were enriched in human EHE. Tumors from our mouse model showed significant enrichment of the human EHE gene set. Finally, using gene-set-enrichment analysis, we demonstrated that both human EHE and murine EHE tumor cells were enriched in YAP/TAZ transcriptional target genes.Two recent studies have generated genetically engineered mouse models of EHE, which conclusively demonstrated that TC is sufficient to generate EHE in vivo. In our study, Seavey et al. generated a conditional knock-in mouse model in which the wild-type ombinase . As the Wwtr1–Camta1 allele are necessary for the generation of tumors. This could be explained either by a gene dosage effect, whereby increased levels of the Wwtr1–Camta1 transcript are necessary to generate tumors in mice, or by a dominant-negative effect, where wild-type TAZ functionally suppresses the action of TC in vivo and inhibits tumorigenesis. We also found it particularly interesting that the ubiquitous conversion of the Wwtr1 locus to a Wwtr1–Camta1 locus by the expression of Cre from the ubiquitously expressed Rosa26 locus solely produced EHE. As TAZ is commonly dysregulated in cancer, one might expect that the expression of a hyperfunctional form of TAZ may generate multiple different cancer types; however, this phenotype was not observed. We hypothesized that this was due to the high transcriptional activity of Wwtr1 within endothelium, as endothelial cells constitute the top Wwtr1-expressing cells within mice by single-cell RNA sequencing. While endothelial cells, or their precursors, are the cells of origin for EHE, it is uncertain whether these tumors originate from a particular endothelial-cell subtype. In addition to the mature endothelium, there are multiple types of endothelial progenitor cells and trilineage  endothelial stem cells, which are necessary for the maintenance of the vascular system [This work left multiple unanswered questions regarding the origins of EHE. The first question is why two copies of the r system ,90,91. IIn a different study, Driskill et al. generated an overexpression system for TC expression based on a Tet-Off approach . ConcordSimilar to other monogenic cancers that contain a single-driver mutation , targeting the TAZ–CAMTA1-driver oncoprotein either directly or indirectly will likely result in a profound clinical effect ,94,95,96As wild-type YAP/TAZ are regulated both by the Hippo pathway and other signaling pathways directly, one can envision that the upstream regulation of TC may occur via both “Hippo-dependent” and “Hippo-independent” mechanisms. An unresolved controversy is whether the TC protein is regulated by upstream controls that normally regulate YAP/TAZ. If the TC fusion protein is controlled by these regulators, the action of TC could be diminished, either by the inhibition of the negative regulators of the Hippo pathway, or by positive regulators of TC. Despite the initial suggestion that TC is unregulated by the Hippo pathway, recent evidence suggests that Hippo alters the action of TC . In unpuIf LATS kinase activity can restrict the nuclear localization and transcriptional activity of TC, augmenting the activation of LATS would be an optimal target to decrease TC activity. However, LATS is negatively regulated by several upstream protumorigenic signals, and it would likely be important to inhibit these signals in order to restore LATS activity in EHE. In other cancers, the inhibition of upstream negative regulators, including EGFRs, GPCRs, and VEGFRs, increases LATS activity . WhetherA notable example of “Hippo-independent” TC regulation is the mevalonate-synthesis pathway, which appears to positively regulate TC activity . TherefoImportantly, YAP–TFE3 appears to be less regulated by the Hippo pathway than TC, and diminishing the function of YAP–TFE3 via the augmentation of Hippo-pathway activity is likely not a potent therapeutic mechanism ,77. ThisAs previously mentioned, TC is a transcriptional coactivator that interacts with the TEAD family of transcription factors and relies on TEAD activity to alter transcription. Therefore, if a small molecule can be designed to disrupt the TC–TEAD interaction, the transcriptional activity of the entire complex can be potently inhibited . As TC lWe recently published a study that demonstrated that TC upregulates connective tissue growth factor (CTGF), and that the expression of this protein is important for oncogenesis . CTGF biNovel model systems, which have only recently been developed, have greatly enhanced our understanding of EHE and have conclusively demonstrated that YAP/TAZ fusions drive EHE and YAP1–TFE3-fused hemangioendothelioma. These fusions function as dysregulated forms of YAP/TAZ and promote tumorigenesis. However, there are key unanswered questions regarding the cellular origin of the tumors, mechanisms of tumorigenesis, and specific dependencies that facilitate oncogenesis. However, we are poised to use these recently developed and faithful EHE models to answer these questions and many more.TC fusion is the only observed abnormality in roughly 50% of the EHE cases, which makes EHE a “genetically clean” model system to understand YAP/TAZ-mediated oncogenesis. With a growing body of evidence demonstrating the central role of YAP/TAZ dysregulation in promoting tumorigenesis, and especially aggressive tumor phenotypes, targeted therapies that inhibit their functions may yield significant therapeutic efficacy. While these therapeutics are in the early stages of testing, the identification of cancers most likely to respond to these therapies is necessary for adequate clinical-trial design. As EHE samples universally express an activating fusion containing TAZ (most commonly TAZ–CAMTA1), EHE presents a robust system for evaluating these therapies.


Ultrafast singlet fission has the potential to facilitate highly efficient photovoltaics through the multiplication of excitons in organic molecular architectures. Here, we consider the interplay of molecular structure and intermolecular coupling toward enabling ultrafast singlet fission and discuss open questions in the field. Early observations of this process date back more than half a century2 but until recently singlet fission seemed fated to anachronism. Renewed interest in the past decade is largely due to the recognition that singlet fission can potentially enhance solar energy conversion efficiency3.Singlet fission is a photophysical process by which a photoexcited singlet state generates two triplet excited states in proximal chromophores4.Recent progress has been particularly strong toward the elucidation of the key intermediate in the scheme, the triplet pair (TT). Ultrafast and multidimensional laser spectroscopy, sophisticated organic synthesis, and powerful electronic structure calculations have combined to produce a detailed picture of triplet pair formation. Time-resolved electron paramagnetic resonance (EPR) spectroscopy has further carried the investigation of the triplet pair, from theoretical construct to experimental validation in the past half-decade. However, completion of the singlet-fission process requires that triplet pairs become independent species . Determining the rules governing this last step are leading to an unexpected richness of the photophysical tableau of coupled organic chromophores5). Molecules absorb light and produce a singlet excited state shared by several neighbors, in which some charge-transfer (CT) character may reside. Subsequently, two localized excitations both in the triplet state (TT) are born, which enables the S1 → TT conversion in some cases as fast as 100 fs. Lastly, the two triplets transfer among the molecules in the ensemble incoherently to become independent excitons (T + T). The details of each step, as they depend on molecular structure and intermolecular coupling, have begun to slowly emerge.Figure 6. However, it is often the case that in a device other fast competing processes, such as energy and charge-transfer at molecule/semiconductor interfaces, occur from singlet states. This degrades the yield of triplets to lower than 200%, limiting singlet fission usefulness in practical situations7. All the same, the limits of ultrafast singlet-fission raise intriguing fundamental questions, because they may encroach on strong coupling and coherent regimes in molecular aggregates. Currently, the primary questions remaining to understand ultrafast singlet fission are related to how to reconcile classic mechanisms in the molecular photophysics of more familiar excited state processes , with the uniqueness of singlet fission, namely the multiexcitonic nature of the singlet-fission products. Further, ostensibly extrinsic factors, such as the accompanying nuclear motions, have emerged as potentially crucial elements. Below we categorize the vanguard research in terms of the pressing questions that continue to drive researchers to investigate the singlet-fission process.Singlet fission can proceed efficiently even on a picosecond or nanosecond timescale1 → TT rate, via the Fermi’s Golden Rule1. In rare cases, this coupling is especially strong and the rate becomes coupling independent—the adiabatic regime8. An active area of singlet-fission research, both computational and experimental, is modulating interchromophore coupling through covalent dimer design and crystal engineering to test the bounds of nonadiabatic vs. adiabatic regimes and to engender both fast TT formation time and efficient TT dissociation. Although it is desirable to be in the strong coupling regime to quickly produce TT, completion of singlet fission to form T + T could still be hindered because a lowering of the S1 energy is often accompanied by strong coupling, resulting in a thermodynamic barrier9. Further, deleterious pathways from TT back to the singlet excited or ground state can be facilitated10.Under most circumstances, electronic state coupling between chromophores is a critical parameter that dictates the S1. The energy of the CT state can be controlled through molecular structure11, crystal engineering, and the solvent environment12. In a near-resonant situation, S1, CT, and TT states may all be accessible on sub-ps timescales, or mixed into the character of the initially photoexcited state. Ultrafast spectroscopy methods can often selectively probe the various species and resolve how they participate in the ultrafast singlet-fission mechanism.The involvement of charge-transfer (CT) character in the photoexcited singlet is another major factor that has been implicated in ultrafast singlet fission, as it can lead to a facile route to triplet pairs through a so-called “mediated” mechanism13. Discovering and designing molecules with an appropriate exothermicity is a topic that has received considerable attention14. However, determining the rate dependence on exothermicity is more complex than measuring the enthalpy change of the overall transformation because the scheme can be arrested at the intermediate bound TT pair unless appropriate avenues for its separation are provided. Here, the factors involved are both enthalpic and entropic, and it is largely the pathways available toward triplet products isolated spatially that determine the speed of free triplet formation. Most polyacene derivatives with five or more benzene rings have ample enthalpic contributions (>0.1 eV) to undergo fast TT formation with even weak coupling, but the second step of singlet fission is only realized in chromophore architectures where triplets can separate efficiently15. Schemes for introducing many product microstates  are now routinely applied to render TT formation ultrafast17 despite limited exothermicity and to assure efficient T + T production, sometimes on timescales of only a few ps.The process of transforming between TT and the pair of independent triplets is often the rate determining step, and can be controlled through modifying the free-energy landscape between reactant and product. The larger the overall energy offset, or exothermicity, the faster the process tends to be, up to a limiting value1. Initially, the singlet-spin state 1(TT) forms due to the principle of conservation of spin angular momentum during the S1 → TT transformation. However, the population can evolve toward other spin states, including the intermediate spin 3(TT) and the highest spin quintet 5(TT). Which states ameliorate or strengthen the binding of the triplets to each other is largely dictated by the proximity and relative disposition of the pair of chromophores involved18. Time-resolved EPR spectroscopy has allowed researchers to investigate which of the various spin states the triplet pair can access and how the population evolves toward independent triplets. For example, 3(TT) may instigate an internal conversion process to a single triplet19, effectively annihilating one of the triplets of the pair and undermining the process. No such scheme exists for 5(TT), and thus in some cases it leads to unique long-lived EPR signatures and eventually independent triplets. Note, however that the time-resolution of EPR is limited to the nanosecond regime; therefore, these measurements are not a direct probe of ultrafast singlet fission21. Theoretical understanding of this process can currently offer only some qualitative guidance, because it requires various approximations, including limiting cases of strong or weak exchange coupling and high molecular symmetry. Progress in this area will require concurrent development of detailed models alongside incisive optical and magnetic resonance probes capable of tracking population dynamics on appropriate timescales in well-defined intermolecular juxtapositions22.The most commonly accepted model of the triplet pair has a manifold of nine spin sublevels into which population may flow after the first step of singlet fission1 to TT and then to T + T are other limiting factors on the singlet-fission rate. In some cases, modes are predicted to break certain symmetries that make TT formation allowed23. In other scenarios, distance or conjugation modulated by intermolecular motions is suggested to improve the electronic coupling, creating a “gate” geometry at which singlet fission is ultrafast25. Intramolecular or intermolecular motions can also instigate fast singlet fission if there exists an intersection between S1 and TT potential energy surfaces (Fig. 26. The involvement of such vibronic modes in the electronic transformation are implicated in a variety of systems27, possibly circumventing the aforementioned need for strong electronic coupling in the equilibrium geometries of the dimer or crystal. If conditions are right the accompanying modes can be transmitted through the intersection and detected in the product electronic states through impulsive excitation and observation of vibrational coherences in pump-probe experiments29. The field has predominantly been exploring and discovering the influence of molecular motions, and rationally creating systems poised to take advantage of these effects will be a considerable but potentially impactful challenge for future investigations.The nuclear motions that accompany the transformation from SAlthough realizing the practical advantages of employing singlet fission in a solar photoconversion system possesses significant challenges, the foundational principles remain under intense investigation by a variety of scientists because of the rich diversity of effects encompassed. Precisely defined intermolecular geometries and experimental conditions will help to improve opportunities to assess comparative singlet-fission models that are currently under intense debate. In particular, more comprehensive models may need to be developed to understand singlet fission in the context of concurrent or competing processes that occur naturally in the bulk or at materials interfaces. Additional opportunities for triplet pair utility, such as in quantum information, will likely emerge as our understanding of dynamics becomes more complete.


Cholinergic drugs are the most commonly used drugs for the treatment of Alzheimer’s disease (AD). Therefore, a better understanding of the cholinergic system and its relation to both AD-related biomarkers and cognitive functions is of high importance.To evaluate the relationships of cerebrospinal fluid (CSF) cholinergic enzymes with markers of amyloidosis, neurodegeneration, neurofibrillary tangles, inflammation and performance on verbal episodic memory in a memory clinic cohort.1–42 (Aβ42), phosphorylated tau (P-tau), total-tau (T-tau), neurofilament light (NFL), YKL-40, S100 calcium-binding protein B (S100B), and glial fibrillary acidic protein (GFAP). Verbal episodic memory was assessed with the Rey Auditory Verbal Learning (RAVLT) and Story tests.In this cross-sectional study, 46 cholinergic drug-free subjects  were recruited from an Icelandic memory clinic cohort targeting early stages of cognitive impairment. Enzyme activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was measured in CSF as well as levels of amyloid-β42 levels and AChE or BuChE activity (p > 0.05). In contrast, T-tau  and P-tau  levels correlated significantly with AChE activity. Although neurodegeneration markers T-tau and NFL did correlate with each other , NFL did not correlate with AChE  or BuChE . Inflammation markers S100B and YKL-40 both correlated significantly with AChE  and BuChE  activity. A weak correlation was detected between AChE activity and the composite score reflecting verbal episodic memory . LASSO regression analyses with a stability approach were performed for the selection of a set of measures best predicting cholinergic activity and verbal episodic memory score. S100B was the predictor with the highest model selection frequency for both AChE (68%) and BuChE (73%) activity. Age (91%) was the most reliable predictor for verbal episodic memory, with selection frequency of both cholinergic enzymes below 10%.No significant relationships were found between CSF Aβ42 levels.Results indicate a relationship between higher activity of the ACh-degrading cholinergic enzymes with increased neurodegeneration, neurofibrillary tangles and inflammation in the stages of pre- and early symptomatic dementia, independent of CSF Aβ The neuropathological changes in AD include the accumulation of beta-amyloid (Aβ) in plaques and hyper-phosphorylated tau protein in neurofibrillary tangles (NFT), leading to loss of synapses, dendrites, and eventually neurons. The most consistent neuronal loss through the progression of AD is found within the cholinergic system of the basal forebrain  or with positron emission tomography (PET) imaging. In 2018, the National Institute on Aging and Alzheimer’s Association (NIA-AA) , neurodegeneration , neurofibrillary tangles (P-tau) and inflammation  among subjects at the pre-and early symptomatic stages of dementia. The second aim was to explore the relationships of the same enzymes with the loss of verbal episodic memory.In recent years, a paradigm shift has occurred from clinical to biological definition of AD based on n = 165). The cohort was comprised of individuals who had been referred to Landspitali University Hospital (LUH) Memory Clinic over a four-year period. The inclusion criteria for joining the cohort study were: (i) a score between 24 and 30 on the Mini-Mental State Examination (MMSE) , GFAP (n = 1), AChE and BuChE (n = 1) values or blood-contamination in CSF samples (n = 3). Clinical diagnosis of AD was based on the criteria for probable AD dementia defined by the National Institute on Aging-Alzheimer’s Association (NIA-AA) , were measured in the ISO 15189 accredited medical laboratory MVZ Labor P.D. Dr. Volkmann und Kollegen GbR . Levels of NFL , YKL-40 , S100B  and GFAP  were measured in a laboratory at the University of Iceland. All assays had mean Intra-assay CV <10% and Inter-assay CV <15%. The activity of BuChE and AChE in CSF was measured as described in Darreh-Shori et al. . The CSF profile was divided into two categories, abnormal (positive) or normal (negative). The specific cut-off points were established by MVZ Labor P.D. Dr. Volkmann und Kollegen GbR, the laboratory performing the ELISAs. Abnormal values were defined as Aβ42 < 375 pg/ml, T-tau > 445 pg/ml and P-tau > 61 pg/ml.Three different CSF biomarker profiles were created, each based on the levels of one core CSF biomarker   were naturally log-transformed to account for a non-normal distribution. Pearson’s correlations and scatter plots were used for estimation and visualization of relationships between continuous variables. Least absolute shrinkage and selection operator (LASSO) regression combined with stability selection was applied for the identification of stable predictors in multivariable models  was found in characteristics between subjects with abnormally low CSF Aβ42 levels  and those with normal Aβ42 levels . In contrast, statistical difference was found between the CSF T-tau profile groups. Subjects with abnormally high CSF T-tau levels (+) were statistically higher in age (p = 0.02), CSF NFL levels (p < 0.001) and CSF YKL-40 levels (p = 0.02) compared to those with normal T-tau levels (-). The T-tau+ group did also score significantly lower on all neuropsychological subtests reflecting verbal episodic memory (p < 0.01).42, T-tau and P-tau) and clinical diagnosis are visualized with boxplots in p = 0.35) or BuChE activity (p = 0.92) between the Aβ+ and Aβ- profile groups. In contrast, AChE activity was significantly higher among subjects with T-tau+ profile compared to those with a T-tau- profile (p = 0.008). No differences were detected between BuChE activity between the same groups (p = 0.11). The same pattern was observed as well for P-tau, with higher AChE activity found among the positive group compared to the negative one (p = 0.02), and no difference between groups in regard to BuChE activity (p > 0.05). No significant difference was observed in AChE (p > 0.05) or BuChE activity (p > 0.05) between different diagnostic groups.Distributions in cholinergic activity (AChE and BuChE) between CSF profiles (Aβp < 0.05) with each other. GFAP did only significantly correlate with the CSF markers S100B  and NFL . No CSF marker correlated significantly with Aβ42 (p > 0.05). All the CSF markers, except for Aβ42 and the cholinergic enzymes, correlated positively with age (p < 0.05). AChE activity correlated significantly with levels of P-tau, T-tau, YKL-40, and S100B (p < 0.05), but not with levels of NFL . BuChE activity correlated significantly with levels of YKL-40 and S100B (p < 0.01), but not with P-tau , T-tau  or NFL .Pearson’s correlations between the CSF markers, age, and education are presented in r = 0.46, p = 0.001), (B) P- tau , and (C) S100B . Weak correlation was found between AChE activity and (D) YKL-40 levels . As with AChE, BuChE also showed moderately strong, positive correlation with levels of (E) S100B  and (F) YKL-40 .Significant relationships of CSF AChE and BuChE activities with CSF markers from 42, T-tau, P-tau, NFL, YKL-40, S100B, GFAP) and three demographic measures .LASSO linear regression with a stability selection was performed for the identification of a set of variables predicting cholinergic enzyme activity with the highest consistency . VariablNo variables reached the selection criteria for prediction of AChE activity . S100B wS100B was also the measure most often selected into a model (73%) for the best prediction of BuChE activity . The CSFr = −0.34, p = 0.02), while BuChE . The analysis was based on 44 subjects as two subjects did not take the RAVLT test.CSF activity of AChE  showed ale BuChE  did not LASSO linear regression with a stability selection was also applied for identifying a set of variables (CSF markers and demographic variables) predicting verbal episodic memory composite score . Only ag42 levels. In contrast, individuals with abnormally high T-tau or P-tau levels had increased AChE activity compared to those with normal levels. Higher levels of inflammatory markers S100B and YKL-40 associated with higher activity of AChE and BuChE. Interestingly, S100B was also the most reliable predictor of both AChE and BuChE activity in comparison to other CSF markers and demographic measures. Weak association was detected between AChE activity and verbal episodic memory performance, although the enzyme did not prove to be a reliable predictor when other measures had also been accounted for. Overall, these results indicate a relationship between higher activity of the ACh-degrading cholinergic enzymes and increased inflammation, accumulation of neurofibrillary tangles and neurodegeneration in the stages of pre- and early symptomatic dementia, independent of CSF Aβ42 levels.We explored the association of cholinergic enzyme activity with CSF markers reflecting amyloidosis, neurodegeneration, neurofibrillary tangles and inflammation as well as with loss of verbal episodic memory among subjects in a memory clinic cohort. Our results indicated no difference in AChE and BuChE activity between individuals with abnormal and normal Aβin vitro and post-mortem studies. AChE activity is consistently increased in regions around Aβ plaques and NFTs at all stages of the disease, although overall AChE activity decreases in the AD brain  of the basal forebrain . The study was conducted in accordance with the Helsinki Declaration latest revision of 2013. The patients/participants provided their written informed consent to participate in this study.UT, TD-S, JS, and PP contributed to the conception and design of the study. UT and MJ contributed to the collection of data. UT wrote the first draft, performed the statistical analysis, and prepared figures and tables. SL provided guidance on statistical analysis. TD-S, SL, MJ, JS, and PP revised the manuscript. All authors contributed to the article and approved the submitted version.SL is employed by deCODE genetics/Amgen, Inc. The remaining 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. The reviewer ER-V declared a shared affiliation with the author TD-S to the handling editor at the time of review.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.


Background: The application of Kinesio Taping (KT) on the lower extremity of stroke patients can improve the quality of somatosensory information by activating lower extremity muscles involved in postural control. Gait analysis and surface electromyography (SEMG) are valuable in assessing the motor ability of the lower extremities.Objective: This study aimed to investigate the effects of KT therapy on gait and SEMG in stroke patients with hemiplegia.Methods: Twenty-one stroke patients were included in the study. KT was applied to the lower extremities of the hemiplegic side. Quantitative gait parameters were measured by a gait analysis system  and activation of the lower extremity muscles were evaluated by the SEMG  before and after taping. Step length, stride length, pulling acceleration, swing power, ground impact, and energy expenditure were used to evaluate when patients walk as usual. SEMG signals were collected from the anterior bilateral tibialis (TA) and the lateral gastrocnemius (LG). The root mean square (RMS) value was used to assess muscle activity. SEMG signals were examined before and after KT treatment in three different locomotor conditions of the patients: walking at a natural speed, walking with a weight of 5 kg, dual-tasking walking  while carrying a weight of 5 kg. The calculation task was to ask the patients to calculate the result of subtracting 7 from 100 and continuing to subtract 7 from the resulting numbers. Comparisons between two normally distributed samples (before and after KT treatment) were evaluated using the two-tailed, paired Student’s t-test.Results: Stride length , pulling acceleration , and swing power  improved in the hemiplegia side after KT treatment. The RMS value of TA SEMG signals in the limbs on the hemiplegia side decreased after KT treatment during dual-tasking walking carrying a weight of 5 kg .Conclusion: KT treatment is effective in altering gait and SEMG characteristics in stroke patients with hemiplegia. Stroke is a common cerebrovascular event that can result in disabilities including motor, sensory, visual, and cognitive impairments . About 7Current common treatment methods for walking rehabilitation include acupuncture, exercise therapy, physiotherapies such as functional electrical stimulation (FES), ankle-foot orthotics (AFO), and kinesio taping (KT) methods . KT is aKT is widely used in the treatment of stroke patients, but the regulation of muscle activation after KT treatment is still unclear. At present, there have been few studies that have objectively evaluated the therapeutic effects of KT on gait stability in stroke patients . RecentlSurface electromyography (SEMG) has the advantages of non-invasion, real-time, and multi-target measurement, and is a method that has received growing attention due to its ability to quantitatively analyze neuromuscular activity in static and dynamic motion states . SEMG isKT is an effective treatment for pain caused by sports injury or other diseases, such as osteoarthritis, chronic low back pain, chronic skeletal muscle pain, and delayed onset muscle soreness . KT can Twenty-one post-stroke patients with hemiplegia recruited from April 2021 to December 2021 were enrolled in this study. Patients who met the following criteria were included: 1) aged > 30 years; 2) diagnosed with stroke; 3) had a 3–6 months course of stroke; 4) the Brunnstrom stages are at least four; 5) able to walk without assistance; and 6) had foot drop after stroke on the hemiplegic side. Exclusion criteria were as follows: 1) patients with cognitive impairment unable to complete experimental procedures; 2) patients with a history of other neurological diseases or disorders, lower extremity surgery or fracture; or 3) patients exhibiting allergy to KT. At baseline, descriptive variables for each patient, including height, weight and Body Mass Index (BMI), were recorded. All patients were in the middle and late stages of stroke recovery, and their activities of daily living were basically self-care (Barthel index > 60 points). The general characteristics of the subjects are shown in Patients underwent routine rehabilitation, including complete training in the hemiplegic limb, exercise training in walking function. All KT treatments were performed by the same qualified physical therapist on the hemiplegic limb of the patient. The area to be taped was cleaned with an alcohol swab prior to KT application. The patients were placed in a supine position during the taping, with their hip, knee, and ankle joints in a neutral position . A kinesvia a wireless Bluetooth connection. Each parameter was averaged over 20 gait cycles to obtain a representative EMG profile for each muscle. SEMG signals were collected from the TA and LG  device. It is capable of identifying 35 activities and postures and can provide an estimate of energy expenditure when incorporated with basic subject anthropometry  and is cA and LG  under thA and LG .The SEMG signals were processed and analyzed using EMGworks Analysis and MATLAB R2020a . The sampling frequency was 1,200 Hz. The root mean square (RMS) value was analyzed and used to evaluate muscle activity. TA and LG muscles were intermittently activated during the test. SEMG data was collected continuously throughout the test. The signals for each dorsal extension and plantar flexion of the ankle were isolated for further analysis. We combined SEMG data from 20 gait cycles using SEMG analysis software. SEMG synchronization points were subjected to analysis using MATLAB software after noise elimination. First, the raw SEMG signals were digitally zero-phase filtered by a second-order Butterworth bandpass filter (20–450 Hz) and then they were full-wave rectified. Second, the RMS values of the full-wave rectified signals were computed within a 50-ms time window. The baseline SEMG activity was determined to be six times the standard deviation of the first 50 ms of the RMS values, where there was no contraction in the dorsal extension and plantar flexion of the ankle muscles. RMS values greater than the baseline were detected and used to determine the beginning and ending of the dorsal extension and plantar flexion of ankle epochs. The difference between the indices of these values was calculated. The point where the difference between the two indices is greater than 250 ms was identified as the end of an epoch. In accordance with t-test. A p-value < 0.05 was considered statistically significant.All the data was analyzed in SPSS software . Data are reported as mean ± standard deviation (M ± SD). The Shapiro–Wilk normality test was performed to examine the normality distribution of the data. Comparisons between two normally distributed samples (before and after KT treatment) were evaluated using the two-tailed, paired Student’s p = 0.029], pulling acceleration , and swing power  all increased after taping when patients were performing walking activities (Stride length [before: 0.89 ± 0.19 (m); after: 0.96 ± 0.23 (m); tivities .p = 0.030) after KT treatment during dual-tasking walking carrying a weight of 5 kg but not for other modes of walking  for all patients. The RMS of the hemiplegic side TA SEMG signals significantly decreased  in affected limbs, improve calf muscle strength, and improve gait stability when patients walked at natural speed. This result is consistent with previous hypothesis that KT treatment would improve the stride length in stroke patients. Furthermore, we also found the RMS value of the affected side TA SEMG signals were different from LG muscles. The RMS value of affected side LG SEMG signals increased after KT taping, but there was no statistical significance (p > 0.05). The RMS value of the affected side TA SEMG signals decreased (p < 0.05) after KT treatment when the patients performed dual-tasking walking carrying a weight of 5 kg. This result is inconsistent with previous hypothesis that the RMS of hemiplegic side TA SEMG signals would increase and RMS of affected side LG SEMG signals would decrease while walking in different locomotor conditions.In this study, we found that KT could increase stride length, pulling acceleration, and swing power  after KT treatment in stroke patients while performing multiple tasks synchronously. We could speculate that when patients performed multitask walking synchronously, the activation of the affected side TA muscle decreased. We considered that the patient should pay closer attention on multitasking, which affected the precision of the walking movement  and the clinical presentation of stroke is complex . We alsoKT therapy is effective in improving gait ability and muscle function among stroke patients with hemiplegia, KT therapy helps prevent falls and promotes recovery among stroke patients with hemiplegia.


This article aims to explore the impact of the punch structure  on the loading, torque and metal flow during the rotary extrusion (RE) process via finite element simulation (FEM) software. In order to further verify the simulation results, physical experiments were carried out and the microstructure of Mg-Gd-Y-Zn-Zr alloy after RE deformation was characterized and analyzed. The FEM results indicated that increasing the groove number will increase the amount of shear deformation and promote the metal at the bottom of the punch to flow rapidly to the cylinder wall. The increase in the groove depth would continue to reduce the forming load and increase the strain. However, if the groove depth exceeded 6 mm, an excessive flow-velocity difference would be formed, resulting in the formation of folding defects. The time of metal flow from the bottom of the punch to the cylinder wall would be shortened with the increase in flaring angle. Therefore, a groove number of 8, an area ratio of 64.49%, a groove depth of 6 mm and a flaring angle ranging from 7° to 9° were the optimal parameters of the punch structure to form the Mg-Gd-Y-Zn-Zr cylindrical parts via the RE technique. In addition, the cylinder parts could be formed with good quality according to the optimized FEM results. The cylinder wall from inner region to outer region exhibited gradient microstructure owing to the different metal flow and strain during the RE process. High-performance magnesium (Mg) alloys have received considerable research interests due to their desirable properties, such as low density, high strength, easy cycling, etc. in the transportation and aerospace fields ,2,3,4. TThe purpose of severe plastic deformation (SPD) technology is to carry out single or multiple deformation passes on the alloy to achieve high strain, so as to make the alloy have superior mechanical properties. SPD technology makes a significant contribution to grain refinement, and the grain size can reach the micron or even nanometer level. SPD methods not only have advantages in grain refinement, but also have a significant effect on the uniform distribution of the second phase of fine particles and the formation of deformation texture, which can greatly improve the strength and toughness of the alloy. It provides a strong support for the development of strengthening and toughening of Mg alloys. At present, SPD technologies that have been researched and developed include repetitive upsetting-extrusion (RUE) ,9,10, muIn addition, Mg rare-earth (RE) alloys have developed from the Mg-Th system and Mg-Y system to Mg-Gd system, and WE54 and WE43 system alloys are high-strength heat-resistant Mg alloys with mature commercialization so far . RokhlinIn recent years, more researchers have proposed a new SPD method to overcome the disadvantages of the traditional SPD methods requiring multi-passes to obtain large accumulative strain . The newThe shear structure parameters at the bottom of the punch are very important. The determination of the number (area ratio), depth of the groove and the flaring angle are the key factors to whether the RE deformation can be well-formed, and these parameters need to be determined by analyzing the gradient simulation results. Finite element simulation (FEM) can quickly optimize the reasonable die structure, shorten the research cycle and improve the success rate of the experiment . TherefoThe general idea of RE deformation research in this paper is RE-parameters simulation-experimental verification, as shown in In the FEM process, the blank size was 260–100 mm . The inner diameter of the female die was 280 mm, and the diameter of the working belt of the male die was 220 mm. The three-dimensional (3D) model of the die and blank was designed by NX software according to the size, and imported into DEFORM-3D software in stl. file format. The H13 die steel provided by the system was selected as the die, and the Mg alloy material model created by the laboratory was selected as the blank. The blank was meshed by tetrahedral elements; the number of meshes was 100,000, and the number of nodes was 29,351. At the same time, mesh rezoning and volume compensation were selected to reduce the calculation error caused by mesh distortion in the deformation process. The critical relative interference depth of mesh rezoning was set to 0.7. In the simulation process, it was set as isothermal deformation and obeys the von Mises yield criterion. The shear friction model was selected for the friction between the blank and the die, because generally, the shear friction was more suitable for metal volumetric forming, and the friction coefficient was set to 0.3. Sparse solver and Newton–Paphson iteration method are used to calculate the whole process of RE simulation. The material model, boundary conditions and friction contact settings are described in The blank height was 100 mm, the total extrusion stroke was set as 75 mm, the longitudinal extrusion speed was 1 mm/s and the die rotation speed was 0.314 rad/s. As the petal-shaped die would rotate the blank in the process of rotation, to avoid the premature rotation leading to the tearing of the blank by torsion, it was first squeezed 30 mm longitudinally to ensure that the working belt of the punch fully entered the blank, and then the longitudinal feed of the punch and the circumferential rotation of the concave were carried out at the same time between 30 mm and 75 mm. In the simulation, the full stroke reverse extrusion of punch without groove was used as a comparative simulation.The billet temperature was set as 420 °C and the die temperature was set as 450 °C, and the RE experiment was conducted on 1250 t-hydraulic press. The observation samples were cut from the initial alloy and the REed barrel for microstructure analysis, and the sampling positions are shown in 4, 3.5 × 104, 4.3 × 104, 4.8 × 104 N·m, corresponding to the groove numbers of 2, 4, 6 and 8, respectively.The comparison of load and torque of FEM under the gradient of the number of grooves (area ratio) is shown in First of all, it is certain that RE deformation can effectively reduce the forming load compared with traditional back extrusion deformation, which has been confirmed by relevant researchers ,28. SecoThe comparison of strain distribution under the gradient of the number of grooves (area ratio) is shown in Therefore, compared with reverse extrusion, RE can bring greater deformation strain. The deformation dead zone under the original backward extrusion punch is improved. The metal under the punch obtains large strain during deformation through the groove structure and flows to the cylinder wall through backward extrusion, which expands the range of the strain layer of the cylinder wall and improves the overall deformation strain. The groove structure with the number of 8 has the widest strain range and the highest cylinder strain value.The histogram of simulated strain-value distribution of cylindrical parts is shown in In the postprocessing of the simulation, the blank model is set with five marking points. According to the flow trace points of the marking points, the metal flow characteristics in the deformation process of reverse extrusion and RE are analyzed and determined. The 2D flow path of the five marked points under different groove structures is shown in The comparison of forming load, strain distribution and metal flow shows that the increase in the number of grooves is beneficial to deformation. According to the FEM results of 0, 2, 4, 6 and 8 groove gradients, the optimal number of grooves can be determined as eight grooves.In addition, the number of grooves is determined as eight, and the influence of groove depth on deformation is also worth exploring. It is inferred from the common sense that if the groove depth is shallow, the degree of shear deformation will decrease, and if the groove depth is deep, forming defects such as folding and tearing may occur. The following will analyze and determine the optimal groove depth through the gradient simulation of groove depth of 5 mm, 6 mm, 7 mm and 8 mm. The groove depth of the eight-groove structure above is 5 mm, so only 6–8 mm is analyzed and compared below.4, ~5.8 × 104, ~6.3 × 104 N·m, respectively. It can be seen from The variation of the simulated forming load and torque under different groove-depth gradients is shown in The load corresponding to each groove depth is close, but the overall trend remains the same. With the increase in groove depth, the load decreases and the torque increases. It shows that on the basis of increasing the number of grooves, deepening the groove depth can continue to reduce the forming load, and the reduction in forming load is accompanied by the continuous increase in torque.The groove depth has a direct impact on the strain value and distribution. The simulated strain distribution results under the groove-depth gradient are shown in From the point of view of load, the increase in groove depth will continue to reduce the forming load; in terms of strain distribution, the increase in groove depth will continue to increase the average equivalent strain, but the deeper groove depth does not mean it is better. The restriction on the groove depth lies in the metal flow. The increase in groove depth will increase the movement speed and inflow and outflow speed of metal along the circumferential direction. If the speed exceeds the appropriate value, it will have a negative impact on the forming; that is, the generation of folding defects. Using the flow example in 4 N·m; the change of extrusion load and torque after the increase of flaring angle is shown in 4 N·m. According to the comparison, the increase in flaring angle has little effect on extrusion load and torque in the range of 3–11°.It can be seen from The average value of the simulated equivalent strain with the flaring angle of 3° is 4.46 mm/mm. The histogram of the equivalent strain distribution of the cylindrical part after increasing the flaring angle is shown in With the increase in the flaring angle, the average equivalent strain of the cylindrical part increases first and then decreases. In order to find out the reason for this change, the blank is marked and its flow path is tracked. As shown in From the change of forming load and torque, the change of the flaring angle has little effect on it. From the strain distribution, the average equivalent strain will decrease when the flaring angle is greater than 9°. After the point-tracking study of the simulation results, it is found that the increase in the flaring angle will shorten the time for the metal at the bottom of the punch to flow to the cylinder wall, and accelerate the flow of the metal that has participated in the deformation to the cylinder wall. Extrusion forming belongs to equal volume forming, so the outflow of metal will be accompanied by the inflow of metal. The outflow of metal involved in shear deformation as soon as possible will make the metal not involved or indirectly involved in the bottom flow into the groove at the bottom of the punch for shear deformation, which can improve the overall deformation strain and deformation uniformity of the cylindrical part. Combined with the distribution of equivalent strain and the law of metal flow, the angle range of the flaring angle can be 7°–9°.5RE phases and cubic RE-riched phases in our previous study [us study . The iniIn addition, the microstructure of Mg-Gd-Y-Zn-Zr alloy in the inner, middle and outer regions after the RE process are also investigated, as shown in There are two limitations in this paper. First, our friction coefficient is a constant value of 0.3, which depends on the lubricant actually used. However, deformation is a dynamic process, and the friction coefficient should be dynamic . Second,Finite element simulation of a punch structure and its effect on the microstructure evolution of Mg-Gd-Y-Zn-Zr alloy via RE method were investigated in the paper. The structure with a groove number of 8 could reduce the forming load of RE by 28.35% compared with reverse extrusion, and increase the average equivalent strain by 201.5%. The increase in the groove depth would produce a difference in the flow velocity of the metal in the groove. The metal in the left and right areas of the groove could fold when the velocity difference along the Y and Z directions exceeded 4 mm/s, so the appropriate value of the groove depth was 6 mm. The influence of the flaring angle on the deformation was mainly to shorten the time for the metal at the bottom of the punch to flow to the cylinder wall, so as to expand the deformation range and forming uniformity. The most suitable flaring angle was 7–9°. The cylinder parts could be formed with good quality according to the optimized FEM results. The cylinder wall from the inner region to the outer region exhibited a gradient microstructure owing to the different metal flow and strain during the RE process. The effect of RE on the mechanical properties of Mg alloys at different deformation rates will be regarded as further study.


The recent decision to remove race-based calculations of kidney function for candidates on the national waitlist approved by the OPTN Board has set the tone towards a more equitable assessment of prospective transplant and donor candidates . The chaIn greater detail, there remains a disparity in providing equitable access to racial minorities , especiaEvidence is lacking regarding ethnicity and organ donation in Europe. In fact, data collection is not generally undertaken and standardized, based mainly on self-identification or recorded country of birth. Furthermore, the discrepancy between national methodologies limits access to data for various minority groups, which in turn renders not only national, but also gathered European data collection less reliable than and less comparable to what happens in the USA . AdditioIn the UK, non-white ethnic minorities, comprise 11% of the population, 7% of organ donors, 35% of people awaiting a kidney transplant and 21% of people who died on the waiting list , with miDemographic characteristics of donors , recipieRace is a variable often considered in eGFR calculations, with the potentiality to overestimate renal function in Black patients, causing about 16% misclassification of kidney disease stage , and thuAs a result of health inequality, Black and Asian minority people in need of a kidney transplant wait for longer in comparison to their Caucasian counterparts . This haTo possibly meet the organ donor offer, LDKT not only represents the best opportunity of success in terms of definitive renal replacement therapy, but it also allows pre-emptive treatment of kidney failure. Since LDKT is unfortunately a precious resource not available for everyone, educational campaigns aiming to expand living organ donation should target these minority backgrounds, and content related to risks for the altruistic act of donation by Black and Asian candidates should cover topics related to the effects of donor and recipient races on the respective health outcomes.What is then the available evidence on the effect of race on living kidney donors, and the impact on recipients’ outcomes? As previously stated, data on post-donation eGFR might be affected by the formulas used in the calculation, so they remain heterogenous and inconclusive, therefore a more accurate analysis could focus on the percentage change in eGFR or slope eGFR in longitudinal observations  or in a per se, should not be a barrier to increase the living donor kidney pool: on average, 88% of the entire living donor pool of this international cohort are Caucasian, but with the help of the previous mentioned educational campaigns, up to 40% of Black and Asian minorities have proven to be a realistic target to contribute to the living organ donor pool   < 0.001 . There h < 0.001 ; howeverFinally, if we look at what happens to Black kidney transplant recipients, in a recent meta-analysis we demonstrated no significant difference between the 1-year mortality in comparison to Caucasians , as wellIn conclusion, Black deceased donors are more likely to experience CKD compared to Caucasians, mainly in view of the trends present in the general population.This should not be considered a barrier to the expansion of the living donor pool and the possibility to offer LDKT to candidates of Black and Asian minorities should instead be concrete and actively incentivized.The new proposed OPTN/UNOS race-neutral eGFR calculations  might beWe believe that future studies need to focus on how to overcome this barrier in consideration of the current organ donor shortage, to minimize the effect of race in kidney function and provide equitable access to individuals of different socio-racial backgrounds. We also strongly support the omission of adjustment for ethnicity in the eGFR formulas, in agreement with current research looking at new endogenous filtration markers and interventions to eliminate racial and ethnic disparities, supporting consideration in health outcome differences due to health inequalities rather than race.Transplant and Nephrology Societies should favor this new policy change to intervene on the long overdue negative impact of race on eGFR, with the aim to reduce delayed referrals for transplant and delays in qualifying for waiting time and for donor’s eligibility. Equity in health means “equal opportunity”  and thus


The level of the virus at the site of infection in mice is significantly decreased upon treatment with orthotopic PTT, and the transfer of the virus to the brain is significantly inhibited. Furthermore, the survival ratio of the mice three days postinfection is increased by intracranial injection of N+TT‐mCB and laser irradiation. Overall, this work provides a platform for the effective treatment of RABV and opens a new avenue for future antiviral studies.Rabies is a fatal neurological zoonotic disease caused by the rabies virus (RABV), and the approved post‐exposure prophylaxis (PEP) procedure remains unavailable in areas with inadequate medical systems. Although strategies have been proposed for PEP and postinfection treatment (PIT), because of the complexity of the treatment procedures and the limited curative outcome, developing an effective treatment strategy remains a holy grail in rabies research. Herein, a facile approach is proposed involving photothermal therapy (PTT) and photothermally triggered immunological effects to realize effective PEP and PIT simultaneously. The designed photothermal agent (N A facile approach that treats the untreatable rabies virus based on a photothermal agent is designed involving high photo‐to‐heat efficiency and positively charged functional groups. The virus is inactivated by photothermal therapy and photothermally triggered immunological effects which can realize effective post‐exposure prophylaxis and postinfection treatment simultaneously. RABV enters the peripheral nerve at a bite site and is transported to the neurons of the central nervous system (CNS), resulting in severe neuroinflammation and 59 000 deaths annually. As there is currently almost no active therapy strategies against rabies, the clinical stages of rabies encephalitis remain untreatable. Although post‐exposure prophylaxis (PEP) can successfully prevent infection, it is still unavailable in some developing countries. The person who was bit always undergoes simple wound flushing for RABV disinfection, but only viruses on the surface can be inactivated. Viruses inside the bite site remain alive, resulting in considerable potential lethality to humans. Additional novel strategies, such as monoclonal antibody‐based approaches, aptamers and small interfering RNAs, have been proposed as forms of postinfection treatment (PIT) to combat rabies. However, a dominant hindrance to the treatment of rabies is its neurotropic characteristic, with the size exclusion limit of the blood–brain barrier (BBB) rendering the delivery of antiviral molecules and drugs to the CNS inherently problematic. Therefore, developing methods to inactivate internalized viruses by simple treatment and improve PIT remain the holy grail in rabies research.Rabies is a fatal neurotropic zoonosis caused by rabies virus (RABV), which is in the genus annually.1 As t Local heat caused by photothermal effects exerts a biocidal effect by destroying the membrane of pathogens, leading to an increase in its permeability. Because of these unique features, PTAs have been used in wearable devices and in vivo virus inactivation. For example, Cai reported a strategy involving binding neutralizing antibodies onto the surface of a photothermal NP to actively capture SARS‐CoV‐2 and kill the virus upon red‐light irradiation. Recently, the PIT of RABV based on PTT has achieved breakthrough results by Zhao et al., using aptamer‐conjugated RVG‐Apt‐PEG‐silica gold nanorods to target RABV in the brains of mice to treat infected mice, which played a guiding role in the treatment of neurotropic viruses. However, the use of multiple injections and irradiation times increases the complexity of PEP treatment procedures. Simplifying the treatment procedure and improving the therapeutic effect are major challenges for rabies PEP and PIT.Photothermal therapy (PTT) utilizes photothermal agents (PTAs) to absorb light energy and convert it into heat, and this method has been widely used in tumor treatment, antibacterial and antivirus research due to its advantages of deep penetration, significant efficacy, low toxicity, and few side effects.6 Loca Inflammatory factors such as tumor necrosis factor alpha (TNF‐α), interferon‐γ (IFN‐γ), and interleukins (ILs) can affect antigen presentation and play an important role in natural immunity. Studies have shown that RABV strains can prevent the entry of immune factors by regulating the permeability of the BBB, while mild inflammatory reactions can effectively eliminate a portion of RABV in the CNS. Overexpression of TNF‐α in recombinant RABV can cause T‐cell infiltration and microglial activation through brain inflammation improving the survival rate of mice. Therefore, the use of a photothermal‐induced immune response to achieve synergistic antiviral effects, which can result in a simplified treatment process and reliable treatment outcomes, has attracted our interest.It has been reported that photothermal effects can induce the release of inflammatory cytokines and immunogens at the irradiation site, contributing to immune stimulation.10 Inf+TT‐mCB nanoparticles) featured positively charged functional groups and high photo‐to‐heat efficiency, which are favorable for RABV targeting and inactivation. The survival ratio of RABV‐infected mice increased after treatment with orthotopic PTT through the inhibition of local viruses via thermal effects and inflammatory factors, including TNF‐α, IFN‐β and ILs ‐absorbing organic PTAs relied on the construction of strong donor (D) and acceptor (A) units into coplanar structures to enhance the non‐radiative decay in the aggregate state. To further elevate the photothermal conversion efficiency (PCE), molecular rotors were grafted into NIR‐based PTA owing to the boosted nonradiative decay by activated intramolecular motions. These strategies have yielded marked success; however, there is still an urgent need to develop a more efficient PTA for more efficient treatment.To improve the therapeutic effect, it is essential to develop high‐performance PTAs, which are photothermal therapy's cornerstone and critical components. Inorganic nanomaterials such as carbon nanomaterials and metal nanomaterials, have been tremendously utilized as PTAs.14 In +TT‐mCB) comprises of three key elements. First, to drive the absorption to the near infrared region, a strong electronic acceptor benzobisthiadiazole (BBTD) with a large planar π‐conjugated structure and electronic donor triphenylamine (TPA) were selected as the molecular skeleton. The planar thiophene (TP) unit was utilized as a π‐bridge to facilitate the intramolecular charge transfer from TPA to BBTD. Second, the long‐branched alkyl chains at the meta position of thiophene provided some necessary room to promote free intramolecular motion for enhanced nonradiative decay. Third, a cationic trimethylammonium unit was introduced to boost D–A interactions for further enhanced nonradiative decay (heat generation). Meanwhile, the cationic units are beneficial for improving the interaction with negatively charged enveloped viruses through charge interactions. The synthetic routes to N+TT‐mCB and other involved molecules are presented in Figure Herein, we proposed a highly efficient PTA designing strategy: introducing ionic groups into strong D–A systems with molecular rotors. As shown in Figure +TT‐mCB, NTT‐mCB without a positive charge was used as a control. The electronic structures in the ground state (S0) determined at the B3LYP/6G(d) level of the theory suggest that both NTT‐mCB and N+TT‐mCB exhibit a planar structure with a dihedral angle between BBTD and TPA of ≈1°  between highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) for NTT‐mCB and N+TT‐mCB  was beneficial for strong absorptivity in the NIR biological window. The slightly larger ΔEgap of N+TT‐mCB was owing to the weakening of the electron‐donating ability of TPA by the electron‐deficient trimethylammonium unit.To better embody the photophysical properties of N° Figure 2. Their2.2+TT‐mCB and NTT‐mCB were characterized by UV–vis–NIR spectroscopy. As shown in Figure +TT‐mCB and NTT‐mCB displayed strong NIR absorption in the spectral region of 600–1000 nm with peaks located at 810 and 838 nm, respectively, which demonstrated a huge potential for NIR light‐triggered photothermal theranostic. The blue‐shifted maximum absorption of N+TT‐mCB compared with NTT‐mCB is mainly owing to the introduction of an electron‐deficient trimethylammonium unit that weakens the electron‐donating ability of TPA, giving decreased D–A effects. Moreover, the larger ΔEgap of N+TT‐mCB also explained its blueshifted wavelength. To analyze these materials in the aggregate state, the NPs were prepared by nanoprecipitation using 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy‐(polyethylene glycol)‐2000] (DSPE‐PEG2000) as the encapsulation matrix. Similarly, the absorption peaks of N+TT‐mCB NPs (≈810 nm) were at shorter wavelengths than those of NTT‐mCB  for 5 min. As shown in Figure +TT‐mCB NPs gradually increased in a concentration‐dependent and time‐dependent manner. The maximum temperatures of N+TT‐mCB after 5 min increased to 75.5 (0.2 mg mL−1) and 72.6 °C (0.1 mg mL−1), better than that of NTT‐mCB . The increased photothermal conversion ability of N+TT‐mCB NPs was attributed to the enhanced nonradiative decay for heat generation owing to the introduction of cationic trimethylammonium unit. The PCE was calculated to ≈78%, which is comparable to other reported organic PTAs  and dynamic light scattering (DLS)  Figure . Further2.3+TT‐mCB NPs were encapsulated by DSPE‐PEG2000, the zeta potential of N+TT‐mCB NPs remained at +17.67 mV  for 0, 3, or 5 min. The experimental process is shown in Figure +TT‐mCB was equally effective in killing the virus against a different strain. Similarly, the virus titer in the cell culture supernatant decreased significantly after 3 min of irradiation, and we could not detect the virus inside the RABV‐infected cells after 3 min of irradiation . The N+TT‐mCB NPs treatment groups were immediately injected with 0.2 mg mL−1 N+TT‐mCB NPs and irradiated with NIR laser  for 5 min. The twice‐irradiated group was irradiated for 5 min again 24 h later without NPs injection. The survival rate of the mice was increased in the treatment group and reached 50% after two rounds of irradiation, while all mice in the control group died  were significantly lower than those in the control group. Notably, the virus was detected in only one mouse on the 8th day. Overall, these results show that the virus can be actively killed at the infection site and that the virus transfer to the brain was reduced through one N+TT‐mCB NP injection and two laser irradiation treatments. The simple operation and effective prevention provide new ideas for the PEP of RABV.Then, to clarify the reasons for the increased mortality, we first quantitatively analyzed the RABV load in different tissues. Hindlimb muscle tissue and brain tissue were collected on different days and subjected to qPCR and virus titer measurements. The qPCR results showed that the viral load at the site of infection was significantly reduced after Ne Figure . Importa+TT‐mCB and laser irradiation, which is favorable for virus inactivation  analysis  can activate astrocytes, which results in the release of angiogenic factors, thus increasing BBB permeability, affecting the passage of cytokines and chemokines and regulating immune responses. Therefore, we conducted a therapy experiment with mice inoculated with 3 × 105 FFU RABV‐CVS11 virus in the hind limb muscles. N+TT‐mCB was injected intracranially after 3 days of infection, and the mice were subjected to 5 min of laser irradiation . Surprisingly, the survival rate was increased by 40% in the N+TT‐mCB + laser group compared with the control group. Clinical symptoms of the mice were observed daily, and the results showed that the clinical score of the mice in the treated group was lower than that of the mice in the control group  for 5 min. We assessed the safety by monitoring body weight changes and performing routine blood analysis, measuring biochemical indexes and analyzing tissue sections. The results showed that the body weight  with strong NIR absorption, high PCE and effective antivirus ability by introducing strong π‐conjugation, molecular rotors and hydrophilic positive charges. By applying the principle of mutual attraction of positive and negative charges, the NPs capture the virus and further kill the virus via PTT. Because of the high antiviral ability, we proposed a simple and effective PEP strategy involving immediate injection of N+TT‐mCB NPs at the site of infection followed by irradiation. The survival rate of RABV‐infected mice increased by inhibiting local viruses via thermal effects and photothermal triggered inflammatory reactions, including TNF‐α, IFN‐β and ILs. Interestingly, when the virus was transported to the brain, the mice were protected by intracranial (i.c.) injection of N+TT‐mCB and irradiation. Overall, the results suggested that N+TT‐mCB + irradiation can serve as not only an effective PEP strategy but also a postinfection treatment method. The photothermal therapy proposed in this study provides a potential method for future antiviral research.In summary, we designed a novel PTA . IL‐6 monoclonal antibody . All animals were used according to the protocol of the Scientific Ethics Committee of Chinese laboratory animals. The agreement was approved by the Animal Welfare and Ethics Committee of the Institute of Veterinary Medicine of the Changchun Veterinary Research Institute .Viruses  were preserved in the authors’ lab. The virus was propagated, titered by N2a cells and stored in a −80 °C refrigerator. N2a (mouse neuroblastoma) and BSR (cloned from BHK‐21) cells were cultured in DMEM  supplemented with 10% fetal bovine serum , 100 units/mL penicillin and 100 µg mLVirus titration assays were performed using N2a cells in a direct fluorescent antibody (DFA) assay. In brief, N2a cells were incubated with tenfold serial dilutions of the virus supernatant at 37 °C for 48 h in a 96‐well cell culture plate in quadruplicate. The cells were fixed with 80% cold acetone for 30 min at room temperature and then washed three times with phosphate‐buffered saline (PBST). Then, the cells were stained with FITC anti‐rabies monoclonal globulin (1:200) and Evans blue (1:500) for 1 h at 37 °C and washed three times with PBST. The positive spots were observed by fluorescence microscopy and the Karber method was used to calculate virus titers.−1 N+TT‐mCB NPs and the infected cells were incubated with 150 µL of DMEM containing N+TT‐mCB NPs . They were irradiated with NIR light  for 0, 3, or 5 min. Subsequently, the irradiated supernatants and the infected cells were separately collected for the detection of virus titers by TCID50 assay. Fluorescence was observed under a fluorescence microscope .BSR cells were cultured in 96‐well plates and infected with CVS11 and SRV9‐eGFP at MOI = 0.1 for 1 h at 37 °C. Then, the liquid was discarded and fresh medium was added to continue the culture for 24 h at 37 °C. After 24 h of cultivation, the cell supernatant was added to the same volume of 0.2 mg mLn = 10) were randomly divided into 3 groups  and inoculated with PBS and N+TT‐mCB by intramuscular (i.m.) injection, respectively. The irradiation groups were irradiated with NIR  for 5 min at the injection site. The twice‐irradiated group received the same treatment 24 h later. After irradiation, the muscle tissues were removed for H&E staining and IHC. Mouse body weights were recorded every other day for a total of 15 days. The blood and organs of the mice were collected 15 days after irradiation. The blood was used for the routine blood analysis and measurement of biochemical indexes. Organs  were fixed for H&E staining and IHC. Moreover, six‐week‐old female ICR mice were inoculated with PBS and N+TT‐mCB NPs by intracranial (i.c.) injection and irradiated with NIR  for 5 min. The brain tissues were removed for H&E staining or IHC immediately after irradiation and 15 days after irradiation, respectively.Six‐week‐old female ICR mice (n = 22) were randomly divided into 3 groups  and were i.m. injected with 5 × 105 FFU RABV‐CVS11. The N+TT‐mCB NP treatment groups were immediately injected with 0.2 mg mL−1 N+TT‐mCB and irradiated with NIR  for 5 min. The twice‐irradiated group received the same irradiation 24 h later without NP injection. The clinical symptoms and mortality were observed once a day for 20 days. The evaluation of clinical symptoms adopts the scoring system, that is, 0 indicates no symptoms; 1 point indicates uncoordinated movement; 2 points indicates rough coat and arched back; 3 points represent tremor and mania; 4 points represent ataxia and paralysis; and a score of 5 indicates death. Mouse brain and muscle tissues were separately collected at 1, 2, 5, and 8 days after irradiation (n = 3) and were used to detect the viral load as well as the expression of inflammatory factors.Six‐week‐old female ICR mice (n = 10) in 2 groups  were inoculated with RABV‐CVS11 at 3 × 105 FFU by i.m. injection. At 3 days postinfection, the N+TT‐mCB group was inoculated with 0.2 mg mL−1 N+TT‐mCB by i.c. injection and irradiated with NIR  for 5 min. The clinical symptoms and mortality were observed once a day for 20 days.Six‐week‐old ICR mice (t test. Values of *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001 indicated statistically significant differences. All data were analyzed using GraphPad Prism 8.All the quantitative data in each experiment are presented as the mean values ± SD. The sample size is provided separately in the figure caption. The statistical significance was determined using Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.