High endothelial venules (HEVs) are specialized postcapillary venules that specifically serve to recruit circulating lymphocytes to secondary lymphoid organs (SLOs) where cognate antigens can be encountered, and immune responses can be initiated. The presence of HEV-like vessels in primary human solid tumours and their association with lymphocyte infiltration and favourable clinical outcomes and response to immunotherapy have provided a rationale for therapeutically inducing these vessels in tumours for immunotherapeutic benefit. Here we specifically discuss evidence for a link between T-cell activation and development of useful tumour-associated HEV (TA-HEV). We discuss the molecular and functional features of TA-HEV, highlighting the benefits for promoting tumour immunity and the important unanswered questions that need to be addressed before TA-HEV induction can be optimized for immunotherapeutic benefit.  HEVs are readily detected by staining with the MECA-79 antibody which recognizes the 6-sulpho sialyl Lewis X epitope presented on peripheral node addressin (PNAd) [Spontaneous formation of TA-HEVs has largely been observed in murine tumours which express strong antigens such as ovalbumin (OVA)  or largen (PNAd) . PNAd isn (PNAd) . Peske an (PNAd) . Interesn (PNAd)  suggestin (PNAd) . For insn (PNAd) .in situ. Factors such as tumour immunogenicity or underlying vascular differences may hold important clues to why TA-HEVs form in some tumours but not others. For example, tumours arising in vivo from injected cells lines are more angiogenic and comprise morphologically immature vessels that have been shown to be more sensitive to vascular disruption in response to inflammatory cytokines such as tumour necrosis factor (TNF) [in situ. Immune activation can also enhance TA-HEV formation in cell line-derived tumours. For example, 4T1 tumour cells inoculated in the mammary fat pad of Balb/c mice were shown to spontaneously form TA-HEVs in approximately 18% of mice. Inactivation of Treg through targeting of the PI3Kδ pathway lead to the majority of treated mice (89%) forming TA-HEVs with higher TA-HEV areas reported in regressor tumours [Indeed, in a mouse model of carcinogen-induced fibrosarcoma, the depletion of regulatory T cells (Tregs) was found to drive the development of TA-HEVs which were associated with reduced tumour growth and increased tumour infiltrating lymphocyte (TIL) frequencies , 19. Immor (TNF) . Neverth tumours . Recentl tumours . Overall tumours . In poor tumours . Further tumours , further+ splenocytes can be recruited to spontaneously induced TLS containing TA-HEVs in a model of inflammation-driven carcinogenesis [–/– mice, which lack all peripheral LNs, leads to the recruitment and induction of specific T cell responses in a B16 tumour model, suggesting in situ priming at TLS sites [What roles might TA-HEVs play in the context of anti-tumour immunity? As in SLOs, several studies have implicated TA-HEVs as active sites for immune cell recruitment. For example, intravenously injected GFPogenesis . FurtherLS sites . In B16-LS sites . SimilarLS sites . Whilst LS sites . With thet al. demonstrated by intra-vital 2-photon imaging that TA-HEVs constitute the major sites of lymphocyte arrest in the microvasculature [− effector/effector memory T-cells and CD62L+ naïve and central memory T cells in short-term homing assays [Only recently have TA-HEVs been shown to directly act as the main sites for immune cell recruitment in three separate tumour cell line models . By inocculature . This waculature . This waculature . Overallculature . Indeed,culature  while hiculature . Indeed,culature  that werculature . TA-HECsculature . TA-HECsculature . Howeverculature . For exag assays . This dig assays . TA-HECsg assays . Interesg assays . Overallet al. examined the impact of LTβR agonist treatment on in-vivo tumour growth using an agonistic anti-LTβR mAb (CBE11) [in vitro suggesting that the anti-tumour mediated effects may not be related to induction of TA-HEVs [The LTβR pathway has been implicated in early SLO development  and in t (CBE11) . Using x (CBE11) . CBE11 w (CBE11) . However (CBE11) . CBE11 w TA-HEVs .et al. showed that LTβR agonist treatment in vitro had no impact on tumour cell proliferation or death [in-vivo experiments revealed that LTβR agonist significantly increased the number of TA-HEVs per tumour area in RT2-PNET tumours but not in MMTV-PyMT tumours [+ T cells around tumour vessels was also highest in this triple therapy group. This was accompanied by an increase in apoptosing cells and necrotic areas which was further enhanced by addition of the LTβR agonist [et al. examined the impact of LTβR agonist treatment on the extent and quality of the T-cell infiltrate in the MCAprog tumour model [−/− mice suggesting the direct activation of LTβR on tumour endothelial cells irrespective of the presence of immune cells [+ T cells [highCD8+ exhausted T cells and terminally exhausted CD8+ T cells and an increase in the proportion of stem-like CD8+ T cells was seen. This suggests that LTβR agonist treatment leads to an alteration in the recruiting functionality of TA-HEVs favouring the recruitment of stem-like T cells. Whilst a significant reduction in tumour weight was reported, MCAprog tumour regression was not reported [+ tumour infiltrating lymphocytes (TILs) which were accompanied by improved responses to therapy and tumour regression [+ human tumour sections from six treatment-naive luminal, Her2+, and triple-negative breast cancer patients were also shown to have stem-like T cells surrounding TA-HEVs [More recent studies have assessed the impact of LTβR agonist treatment and its relation to murine tumour growth and TA-HEV formation  and administration of LTβR agonist , 13, 35 aneously . It is integrity . Furtherntegrity . CD4 T cntegrity . Overallntegrity . Howeverntegrity .+ vessels (which otherwise do not form spontaneously) and significantly more CD3+ immune cells per tumour surface area [+ T cells per tumour surface area and extent of granzyme B (GrzB) expression was comparable to that of untreated tumours [+ T cells and an upregulation of GrzB expression [+/GrzB+ effector T cells and reduced the metastatic burden significantly [This synergism between LIGHT delivery, vessel normalization and immune activation via ICB (PD-L1 blockade) was further demonstrated in a mouse model of GBM . As withace area . However tumours . Only whpression . Importapression . While Lpression , 46 , thpression . LIGHT-Vpression . Interesficantly . Howeverficantly . Consistficantly .et al. first demonstrated that the majority of arising TA-HEVs do not come from clonally expanding ECs but rather via post capillary venule (PCV) metaplasia [+ aggregates and tumour relapse [Taken together it is clear that LTβR agonist or LIGHT monotherapy whilst sufficient in inducing TA-HEVs (in some mouse tumour models) is largely insufficient in driving effective anti-tumour immune responses. Furthermore, the maturation of TA-HEVs in response to immune activation following ICB in the MCAprog model , supporttaplasia . This artaplasia , 49 . Im relapse . This su1β2 lymphotoxin production by CD8 T cells and NK cells were shown to drive TA-HEV formation via signalling through the LTβR/noncanonical NFkβ axis [α3 by effector lymphocytes and signalling through TNFRs was shown to drive PNAd expression [How might immune activation promote the formation of TA-HEVs? Several factors released by immune cells have previously been shown to induce TA-HEVs. In the PyMT tumour model, LTαFkβ axis . In contpression . In Tregpression . Interespression , 18. Ovepression  is dispeRecent efforts have shed light on how TA-HEVs arise  and the


In a porous medium, we have examined sinusoidal two-dimensional transport enclosed porous peristaltic boundaries having an Eyring Powell fluid with a water containing  Peristalsis is a transport generated by the wave-like contraction and expansion of a duct-containing fluid. If there is no peristaltic movement then food bolus cannot be pushed through the digestive tract, urine cannot be moved through the urinary duct and spermatozoon, ovum, embryos and eggs cannot be moved and transferred into a reproductive duct. A person faces diarrhea or constipation in the absence of peristaltic motion. Also, the movement of lymph through lymphatic vessels and the motion of capillaries, arterioles and venules is peristaltic. Peristaltic pumping also acts as a cleansing agent that removes bacteria and gas and controls bacterial growth in large intestines. The peristaltic mechanism is applicable in engineering and biomathematics like the design of finger pumps and roller pumps, dialysis and blood pump machines.1 and further work on this topic is proceeded by Latham2 in detail as Fluid motions in Peristaltic Pump in MIT-Press, Cambridge. There are many investigations studied mathematically for Newtonian and Non-Newtonian fluids. Since there is a vast use of fluids like water, air, glue, paint and blood in our daily life, this domain of research has sought the attention of researchers.The First theoretical and experimental study on the topic of peristalsis in which the author derives the relation between motion of fluid and the amplitude ratio of the peristaltic transport has been done “Peristaltic transport”, by Fung and Yih3 “Peristaltic transport” in which he uses a sinusoidal channel to describe the transport of mass and finds the mean average velocity of the transport and4 he study about arbitrary Reynold number, wave number and amplitude ratio for peristaltic pumping. Srivastava et al.5 worked on the perturbation solutions of peristaltic flow in a channel filled with Newtonian fluid for a small amplitude ratio. Husseny et al.6 have looked into the Effects of porous boundary walls on peristaltic walls through a porous medium”. “The flow of Maxwell fluids in porous media and peristaltic domain” is examined by Whitaker et al.7. Moreover, Al-Arabi et al.8 also show their interest in the domain of peristaltic flow and explored Non-linear peristaltic channel of Magnetohydrodynamic flow. Further “Peristaltic Flow of a Maxwell fluid Mode through Porous Boundaries in a Porous Medium9 and Corrugated walls analysis in microchannels through porous medium under Electromagnetohydrodynamic (EMHD) effects10 has been examined by Nadeem et al. The peristaltic channel has scientific contributions in industrial and biomedical applications and drug delivery.Jaffrin and Shapiro have done many investigations like10 has been studied the characteristics of heating scheme and mass transfer on the peristaltic flow for an Eyring-Powell fluid in an endoscope. Moreover, Khan11 analyzed the Analysis of eyring–powell fluid flow used as a coating material for wire with variable viscosity effect along with thermal radiation and joule heating. Also Riaz and Ellahi studied Role of hybrid nanoparticles in thermal performance of peristaltic flow of Eyring–Powell fluid model12. In this subclass of fluid which deals with Eyring Powell fluid there is not much research but all research in this subclass is not mentioned here. The Eyring Powell fluid has significant impact on the field of fluid mechanics like in viscosity prediction, reaction kinematics, molecular diffusion, polymer science and lubrication technology.Eyring Powell fluid is a non-Newtonian viscoelastic fluid which has been studied by Eyring Powell in 1944. Although Eyring Powell fluid is discussed by some researchers but still it is not very common in the area of research. Akbar and Nadeem11 studied the “Role of hybrid nanoparticles in the thermal performance of the peristaltic flow of Eyring–Powell fluid”. Moreover, McGrail et al.13 studied Metal–organic heat carrier nanofluids. Transport properties of non-Newtonian nanofluids and applications is studied by the Sivaraj et al.14. Also, Naveen Kumar analyzed non-Newtonian hybrid nanofluid flow over vertically upward/downward moving rotating disk in a Darcy–Forchheimer porous medium15.The Nanoparticles like metals, oxides, carbides, 6 is studied by Shehawey and Husseny. Nonlinear peristaltic transport of MHD flow through a porous medium was investigated by Mekheimer8. Further, Peristaltic flow of a Maxwell fluid in a channel with compliant walls is discussed and analyzed by Hayat17. Also Alame18 take part in the research of this field by using uniform magnetic field in the channel having non-Newtonian Fluid with heat and mass transfer through a porous medium. Further, Peristaltic flow of phan-thien-tanner fluid has been studied by Vajravelu et al. in an asymmetric channel with porous medium19 and also Peristaltic flow and heat transfer of a Herschel-Bulkley fluid in an inclined non-uniform channel with wall properties is also done by Vajravelu et al.20.Fluid for a permeable medium has a broad scope in filtration, ground water flow, biomedical applications and importance in engineering and fluid mechanics. In our daily life, permeable media includes wood, foam, and rocks. Industrial and domestic applications of a thin permeable layer are cells, batteries, filters and printing papers. Permeable media had drawn the attention of many researchers such as the “Effects of porous boundaries on peristaltic transport through a porous medium”The aim of the study is to check the mathematical behavior of non-Newtonian viscoelastic Eyring Powell Nanofluid having incorporated Consider an infinite 2d channel of thickness 2b, filled with an incompressible viscoelastic Eyring Powell Nanofluid containing mentclass2pt{minimS for Eyring Powell fluid is:The equations which govern the incompressible flow is:6 and energy equations from18.The fluid in a porous medium has the resistance:For solving equations, we make stream function kes Eqs. , 6) and and\docukes Eqs.  and reduBy putting the below non-dimensional parameters in Eqs. –13)14\d14\d13)1410. The term 6.Solution of To find the solution of Eq. , we appl6. As we know that the fluid enters the flow region through one plate at the same rate as it exits the other plate with velocity V so, the solution of Here we have used the same solution technique used by et al. HussenyIt should be noticed that the present problem is not an Eigenvalue, and the boundary conditions are not all homogenous as in all problems of hydrodynamic stabilities. So, to solving Solutions of 18 is satisfied by The following type of solution according toing Eqs. –34) in  in 18 ising Eqs. –24) we  we 18 isBy solving , 36) an an36) anBy using –41) in  in 42\do) in Eq. , we get\Numerical computation is done to comprehend the impact of the channel’s porosity and the medium’s permeability parameter on the mean-time average velocity. A detailed examination of Eq.  reveals The Eqs.  and 46)46) deter8.The value of 8 has plotted graphs of u for The for Eq. . For graentclass1pt{minimaV, M and W. Figure V rises the constant M and the permeability parameter W.In Figs. entclass1pt{minimaV, M and W. It is observed in Figs. V at the boundaries, and permeability parameter W while rises with the rise in the Eyring Powell Parameter M.In Figs. M is inversely proportional to the viscosity so by increasing viscosity, M decreases which means fluid becomes more viscous and hence velocity decreases. The value of constants According to Figs. M is greater, fluid will be less viscous, and velocity will be greater and vice versa. According to this relation, overall velocity should have to rise in the whole channel by increasing M but in the upper portion, there is variation in behavior due to the impact of suction. According to Fig. W rises means that more fluid can pass through pores with the rise on permeability.Figure y and reflect that by increasing porosity V of the boundaries and volume fraction V of the boundary’s temperature  Figures mentclasspt{minimaIn Figs. V, while M and Permeability W.The dominant constant W and porosity V while rises with the rise in the Eyring Powell parameter M.The constant The Velocity drops with the rise in the viscosity but in the upper segment of channel suction disturbs this behavior.The rise in permeability raises the velocity of the flow means it allows more fluid to pass through the medium.The Velocity rises with the rise in the Eyring Powell parameter in lower segment of channel and drops in upper segment of the channel.The rise in the porosity of the wall causes the drop in the mean velocity of the lower segment of the channel and rises in the upper segment of the channel.Temperature The rise in the volume fraction In this study, we have talked about an Eyring Powell fluid model with Data availabilityThe authors states that all the files are provided in the paper no hidden file is required however if journal required any further data from us we will provide and the corresponding author is responsible to provide to the journal.


Induced by varicella zoster virus (VZV), postherpetic neuralgia (PHN) is one of the common complications of herpes zoster (HZ) with refractory pain. Animal models play pivotal roles in disclosing the pain mechanisms and developing effective treatments. However, only a few rodent models focus on the VZV-associated pain and PHN.To summarize the establishment and characteristics of popular PHN rodent models, thus offer bases for the selection and improvement of PHN models.In this review, we retrospect two promising PHN rodent models, VZV-induced PHN model and HSV1-induced PHN model in terms of pain-related evaluations, their contributions to PHN pathogenesis and pharmacology.Significant difference of two PHN models is the probability of virus proliferation; 2) Most commonly used pain evaluation of PHN model is mechanical allodynia, but pain-induced anxiety and other behaviours are worth noting; 3) From current PHN models, pain mechanisms involve changes in virus gene and host gene expression, neuroimmune–glia interactions and ion channels; 4) antiviral drugs and classical analgesics serve more on the acute stage of herpetic pain.Different PHN models assessed by various pain evaluations combine to fulfil more comprehensive understanding of PHN. Varicella-zoster virus (VZV) is one kind of human neurotropic viruses . PrimaryTo date, two vaccines for VZV infection are available: a live-attenuated vaccine and a new adjuvant recombinant VZV vaccine . HoweverAppropriate animal models are required to explore PHN pathogenesis and support the examination of new prevention and treatment strategies. Although several animal models of VZV infection have been published and recapitulated to help understand VZV biology and HZ onset, there is only a few rodent models focusing on the VZV-associated pain and PHN. In 1999, behavioural changes suggestive of abnormal pain state in VZV-infected rat models was firstly described –12. HoweVaricella-zoster virus (VZV) and herpes simplex virus 1 (HSV1) are human neurotropic alpha-herpesviruses that often acquired early in life . FollowiVZV particles are pleomorphic to spherical in shape, about 150 ~ 200 nm in diameter and are composed of three protein layers: a nucleocapsid containing the viral double stranded DNA (dsDNA) genome, a tegument layer surrounding the nucleocapsid, and an envelope with viral glycoproteins facing outwards . VZV genIn most tissue culture cells, HSV1 is an efficient virus that can overcome the species specificity, whose lytic replication cycle culminates in 8 ~ 12 h. Although the VZV lytic replication cycle culminates in 9 ~ 12 h , it can Upon VZV virion entering the host cell, tegument proteins are released into the newly infected cell and alter the host environment, thereby inhibiting antiviral responses and influencing the virus program, that is, a lytic or latent infection. During productive infection the complete VZV proteome consisting of some 68 unique gene products is expressed through interaction of a small number of viral transcriptional activators with the general transcription apparatus of the host cell. VZV virion proteins delivered into newly infected cells upon entry are not absolutely required to initiate VZV gene expression, as evidenced by the resulting VZV replication upon transfection of cells with viral DNA. It has been reported that protein-free HSV-1 DNA is also infectious when properly introduced into cells, albeit at low efficiencies. However, the relationship between virion gene expression and VZV-associated pain remains unclear.rd day following injection , the increased expression of interleukin-1β in the astrocytes induced N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to strengthen pain transmission . Althoug+ channel Kir4.1 in satellite glial cells and impairing K+ homeostasis 1.3 and Na(v)1.8 sodium channels has been proved in the DRG of the rats, which are thought to be related to VZV-induced neuropathic pain . In HSV1eostasis .Ntrk2, Trpv1, and Calca (CGRP)  , which aUp to 90% of HZ patients experience acute pain, which may be alleviated by timely antiviral administration to limit viral replication . ProvidiCurrent analgesic treatment strategies for PHN include tricyclic antidepressants, topical lidocaine or capsaicin patch treatments, opioids and gabapentinoids .In the HWhile there is strong clinical evidence supporting the efficacy of certain analgesic therapies verified from rodent models, most of these therapies do suffer from a poor translation and narrow therapeutic index which limits their clinical effectiveness , 58.via certain biological processes (vHPPE) was expressed using a herpes simplex virus vector to deliver the gene in rats injected with VZV  has been found to have anti-inflammatory and analgesic effects. Saline with a therapeutic dose of hydrogen  is used to deliver molecular hydrogen  has been identified to be essential for VZV infection and spread through recognizing VZV glycoprotein E  to avoid virus infection, which requires rigorous experiment environment, such as isolated rooms for virus growth and injection . Some PHAs for the pain evaluation in PHN rodent models, it can be more comprehensive in the future, focusing more on the non-stimuli responses after inducing neurological pain. Besides, functional imaging in unanaesthetised PHN models is extremely rare, as most of pain researches in humans derives from imaging studies . DevelopMO: Drafting/revision of the manuscript for content, including writing for content; Analysis or interpretation of data. JC: Drafting/revision of the manuscript for content, including writing for content; Major role in the acquisition of data. SY: Drafting/revision of the manuscript for content, including writing for content; Analysis or interpretation of data. LX: Study concept or design; Analysis or interpretation of data. DX: Study concept or design. SW: Drafting/revision of the manuscript for content, including writing for content; Study concept or design; Analysis or interpretation of data. All authors contributed to the article and approved the submitted version.


This review will cover the recent developments in our understanding of neutrophil heterogeneity in HCC and how neutrophils can be harnessed to improve HCC immunotherapy.Neutrophils, until recently, have typically been considered a homogeneous population of terminally differentiated cells with highly conserved functions in homeostasis and disease. In hepatocellular carcinoma (HCC), tumour-associated neutrophils (TANs) are predominantly thought to play a pro-tumour role, promoting all aspects of HCC development and progression. Recent developments in single-cell technologies are now providing a greater insight and appreciation for the level of cellular heterogeneity displayed by TANs in the HCC tumour microenvironment, which we have been able to correlate with other TAN signatures in datasets for gastric cancer, pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). TANs with classical pro-tumour signatures have been identified as well as neutrophils primed for anti-tumour functions that, if activated and expanded, could become a potential therapeutic approach. In recent years, therapeutic targeting of neutrophils in HCC has been typically focused on impairing the recruitment of pro-tumour neutrophils. This has now been coupled with immune checkpoint blockade with the aim to stimulate lymphocyte-mediated anti-tumour immunity whilst impairing neutrophil-mediated immunosuppression. As a result, neutrophil-directed therapies are now entering clinical trials for HCC. Pharmacological targeting along with  Primary liver cancer is a leading cause of cancer-related death and morbidity, with 905,667 cases and 830,180 related deaths in 2020 . FailureThis review will cover recent advances in our understanding of neutrophil function and heterogeneity in HCC and discuss how this heterogeneous population of neutrophils may impact the TME and their cellular plasticity can be harnessed to improve immunotherapy in HCC.Phenotypical and functional heterogeneity in myeloid cells is well established for monocyte and macrophage populations and assures diverse functions that are required to maintain homeostasis, respond to infection and resolve inflammation . On the Initial transcriptomic studies in 2020  defined Our group and others have suggested that neutrophil maturation plays a critical role in determining their phenotype and functionality. Fundamental to this, is the recently proposed ‘neutrotime paradigm’ which aims to provide a framework to understand and describe heterogeneity in neutrophil populations. In this model, neutrophils, rather than existing in discrete maturation states as described previously, follow a developmental continuum, called the ‘neutrotime’ . As neutFor over a decade, neutrophil heterogeneity in cancer has been dichotomously described with circulating neutrophils labelled based on their density as either high/normal- or low-density neutrophils  and TANs labelled based on either their identified pro- or anti-tumour functions . EmerginSingle-cell transcriptomics has revolutionised our understanding of the tumour microenvironment, providing a wealth of data on nearly all cell types at single-cell resolution. However, until recently, the majority of scRNAseq studies failed to recover a significant number of neutrophils for downstream analysis; this is a feature also prominent in other single-cell analyses performed in solid organs . Reasons+, ISG15+ and TXNIP+ subtypes are found in peripheral blood, the ELL2+ and PTGS2+ mainly in adjacent liver and the MMP8+, APOA2+, CD74+, IFIT1+, SPP1+ and CCL4+ predominantly located in the tumour  signalling and pathways of type I interferon signalling and response to interferon gamma . Th. Th+ T cignature . Interesignature  and a phignature  is curreGiven that neutrophils display a range of anti-tumour functions, we propose that due to their cellular plasticity it may be possible to reprogram TANs to promote anti-tumour immunity and that this may not necessarily be specific to targeting a chemokine receptor as we have demonstrated. Esteban-Fabró and colleagues showed that combining anti-PD-1 therapy with Cabozantinib, a small molecule tyrosine kinase inhibitor largely selective for c-Met and VEGFR2, increased anti-tumour efficacy compared with monotherapies . This thDue to our recent improvements in the understanding of intratumoural neutrophil populations it is now clearer than ever the importance of understanding patient-specific neutrophil heterogeneity. As more neutrophil specific therapies are developed and tested in combination with immune checkpoint immunotherapies, it will be critical to understand how these drugs impact each neutrophil population and how the abundance of each one in the patients' tumours impacts therapy outcome.in vitro and in vivo. Mechanistically, the CAR-neutrophils were shown to be phenotypically similar to hPSC derived neutrophils but had greater anti-tumour functionality as assessed by neutrophil-tumour immune synapse formation, phagocytosis and ROS-mediated tumour killing , which significantly enhances their anti-tumour potential. However, to date, efficacy in solid tumours has been limited , althoug killing  96]. In. Inin vitrophils ,98 and d[in vivo  99]. Fu. Fuin viNASH-HCC . CurrentNASH-HCC . In theotoxicity .Interrogation of TAN biology has finally entered the era of scRNAseq allowing for the first time the analysis of neutrophil heterogeneity and the identification of TAN subtypes, many of which appear to be conserved across cancers. However, this is just the start, and this cutting-edge technology is not without its limitations. Firstly, scRNAseq read depth is much lower than that of conventional bulk RNAseq and, as such, only a superficial picture of the neutrophil transcriptome is provided . When thFrom a therapeutic perspective, neutrophils have conventionally been regarded as promoting tumorigenesis. As a result, immunotherapeutic strategies targeting neutrophils in cancer have largely focused on inhibiting the recruitment of neutrophils to the tumour. Recent data, however, suggest that neutrophils in the HCC tumour microenvironment can be programmed with anti-tumour functions if provided with the right exogenous stimulus and could even overcome immunotherapy resistance in NASH-HCC patients. Finally, neutrophil-based cell therapies may provide an exciting new therapeutic avenue and an area of research for improving our understanding of neutrophil-mediated anti-tumour immunity and improving HCC immunotherapy outcomes. Altogether, recent developments in our understanding of neutrophil biology in cancer has shed light on the cellular heterogeneity and plasticity of a once considered ‘inert’ cell. Future work should aim to exploit these recent findings to boost the efficacy of existent treatments and lead to the development of new immunotherapies for patients with cancer.Neutrophils are a heterogeneous population of cells that can display a range of phenotypes in homeostasis, disease and cancer.Tumour-associated neutrophil (TAN) subtypes functionally correlate between cancers.Neutrophils exhibit cellular plasticity and can undergo reprogramming in the HCC tumour microenvironment.in situ or ex vivo, represent a viable anti-cancer target.Reprogrammed neutrophils,


Our aim of study was to evaluate joint fluid cell count for diagnosis among septic arthritis patients confirmed with positive culture in children.Septic arthritis is one of serious pediatric infections that could result motor comorbidities. Historically, joint fluid cell count of 50,000 and over cells/mmPatients with septic arthritis were included at Tokyo Metropolitan Children's Medical Center from March 2010 to March 2023. Septic arthritis was confirmed with positive joint fluid culture for a pathogenic organism. Patients with negative joint fluid culture and patients without joint fluid cell count were excluded. Electrical charts were retrospectively reviewed for demographic data, timing of arthrocentesis, culture result and joint fluid cell count.3. Patients with 50,000 and over cells/mm3 was 23%. The median time from onset to arthrocentesis was 3 (IQR: 2-5) days. The isolated organisms were methicillin-susceptible Staphylococcus aureus (50%), methicillin-resistant S. aureus (9%) and Streptococcus pyogenes (27%), Streptococcus pneumoniae (5%), Klebsiella pneumoniae (5%) and Salmonella spp (5%). The infected joints were hip (59%), knee (23%), ankle (14%), elbow (5%) and shoulder (5%). One patient had hip and ankle arthritis. Nine patients were complicated with septic osteomyelitis. The Pearson correlation coefficient between days from onset of symptoms to puncture and the number of joint cells was 0.093 (p=0.681). A total of 95 patients with septic arthritis were identified. After excluding patients with negative culture and without joint fluid cell count, 22 patients were included for our study. Median age was 5 (IQR: 2-10) years old. Girl ratio was 45%. The median joint fluid cell count was 19,575  cells/mm3.Majority of patients with septic arthritis confirmed with joint fluid culture did not exceed joint fluid cell 50,000 cells/mmAll Authors: No reported disclosures


Through modelling, the average interstitial velocity was estimated and found to be higher than the corresponding value obtained from the Dupuit–Forchheimer equation. The discrepancy obtained suggested that the flow behaviour within the filter channels differed from that based on the simplified assumptions of the equation. The porosity and permeability of the CFFs were evaluated during the post-processing stage using surface integration and user-defined equations. The experimentally determined porosity closely matched the values obtained from the simulation model, demonstrating the reliability of the numerical approach. However, the permeability values from the simulation of CFFs of grades 30 and 50 were higher than those obtained experimentally. This discrepancy can be attributed to the larger channels in the generated geometrical pattern compared to the original CFF structure. The present findings highlight the effectiveness of the proposed methodology in developing a representative macro-scale geometry for CFFs and in simulating fluid flow behaviour.To investigate the fluid flow characteristics of conventional Ceramic Foam Filters (CFFs) of grades 30 and 50, a 2D macro-scale geometry was generated by converting pixel grid images of the filters into vector format images. The flow behaviour through the filter channels was then numerically modelled using the Stocks equation within the Creeping Flow interface of COMSOL Multiphysics If the interfacial tension between a particle and the collector is lower than the interfacial tension between the particle and the melt, as well as than that between the melt and the collector, the particles are captured by the collector [Non-metallic inclusions introduced during the melting, alloying, and transfer processes in aluminium production are effectively removed before casting by employing alumina (Aling step . The priollector . In the ollector . The comollector . Additioollector .iRe) is less than unity [Aluminium filtration typically occurs at a velocity of approximately 10 mm/s , and thean unity . Understan unity . Additioan unity ,10. The 2O3-based CFFs was empirically evaluated using water as a surrogate due to its similar dynamic viscosity to that of molten aluminium at the casting temperature, i.e., 993–1003 K [2∙s2 or Pa/m), 2), and u is the superficial (seepage) velocity (m/s), which is the flow velocity expressed as the ratio of the volumetric flow rate to the cross-sectional area from which the fluid is flowing.The permeability of Al3–1003 K ,12. As w3–1003 K :(1)−∇P=μNumerical simulations provide an alternative approach to conventional empirical methods because conducting experiments is often expensive and time-consuming. In view of this, the study of fluid flow through porous media has, over the years, been performed using various approaches, including pore-scale generation methodologies and numerical models for computing flow fields within the complex void space of the filter ,16,17,18Regulski et al.  numericaform drag coefficients by simulating the transport properties of flow in a 3D digital representation of a 10 PPI reticulated porous ceramic. The permeability obtained from the numerical simulation was compared to the values predicted by analytical equations, with the two-point correlation bound [Similarly, Carvalho et al.  performeon bound ,20, prov2O3-based CFFs with grades 30 and 50 were generated, and numerical CFD simulations of a water flow through the channels was performed using COMSOL Multiphysics® as the platform for solving the CFD codes. The modelling results were utilised for postprocessing the numerical data to evaluate the permeability of the CFFs.In the present work, 2D geometrical patterns of AlThe flowchart presented in ® (version 5.3). Struts, and the blue regions represent the channels. By converting the scanned images into a vector format, distinct divisions within the image were created due to the colour contrast between the resin and the CFF material. In view of this, the bitmap images obtained from optical scanning were converted to the Drawing Exchange Format (DXF), as shown in Struts were subsequently deleted using the functionalities in the Model Geometry interface, resulting in Commercial CFFs of grades 30 and 50, measuring 584.2 × 584.2 × 50.8 mm, were used to create cylindrical samples. The filter samples were longitudinally sectioned, and half of the sections were impregnated with blue metallographic castable mounting resin using a vacuum mounting apparatus. This impregnation process enhanced the resin penetration into the voids and facilitated the release of any trapped bubbles. The surface of the sectioned sample, parallel to the height, was ground to remove excess resin and then polished to eliminate scratches and, thereby, obtain a smooth surface. A flatbed scanner  with a resolution of 2000 dpi was used to scan the sample surface. The scanned images were processed to recover the geometry pattern of the CFFs for importation into the simulation software, i.e., into COMSOL MultiphysicsParametric Sweep study was performed with a sequence of pressure drops within the region of interest.Volumetric flow rate through the outlet, the Darcy velocity at the outlet and the Permeability were defined as user-defined variables for post-processing evaluation of the permeability. The ® (version 5.3) CFD code was used to solve the incompressible continuity and Stokes equations in the pore space geometry of the CFFs.P is the pressure force (Pa), µ iu is the velocity vector (m/s), F indicates all forces other than the pressure, and ρ is the fluid density (kg/m3). Equations (4) and (5) involve the Laplace operator and the substantial time derivative .2O3) CFF tiles/blocks measuring 58.4 × 58.4 × 5.1 cm3 using a core drill machine, resulting in samples with a nominal diameter of 51 mm. As shown in The CFF samples selected for the investigation were obtained by cutting commercial alumina  and dynamic viscosity (µ) were determined by substituting the measured temperatures into Equations (6) and (7), respectively [A, B and C, with values of −4.5318, −220.57 and 149.39, respectively, were employed for the temperature range of interest (270–380 K).A water loop system was utilised to remove air from the pipes and the CFF samples prior to starting the measurements. Water at a temperature of 283 ± 3 K served as the working medium throughout the experiments. The water density ρTA denotes the dynamic viscosity of water over the intrinsic permeability (kg/m3·s), and Finally, the permeability of the CFF samples was determined using a polynomial interpolation equation of the form arealϕ) of the CFFs was determined by evaluating the ratio of the voids’ area to the total area, according to Equation (8) [By assuming a homogenous structure within the CFFs, the areal porosity (tion (8) . The arevoidsV) over the total volume . The total porosity was determined using the density of the CFF . This equation represents the fraction of the volume of the voids v¯=Jk, see The permeability of the CFFs of grades 30 and 50 was determined during post-processing using the user-defined variable ® to simulate a fluid flow through the filters using the creeping flow interface. This simulation was performed within a flow velocity range where the iRe, calculated based on experimentally determined superficial velocity values, was less than 1. The analysis yielded the following conclusion:The introduced procedure successfully generated a 2D geometrical pattern of CFFs that accurately replicated the tortuous surface path through the CFFs, for which fluid flow could be simulated.The areal porosity obtained through 2D CFD modelling was comparable to the experimentally determined porosity.The CFD modelling revealed that the interstitial velocity, obtained by averaging the instantaneous velocity throughout the macro-structure, was higher than the intrinsic velocity calculated using the Dupuit–Forchheimer equation, which relies on the superficial velocity and porosity of the porous medium, i.e., of the CFF.® were higher than the experimentally obtained permeabilities. This discrepancy was attributed to the non-representative nature of the geometrical pattern employed for the investigated CFFs.The permeabilities obtained from the CFD modelling using COMSOL MultiphysicsA novel method was employed to generate a 2D macro-structural model of CFFs for the CFD modelling of their hydraulic properties. The geomaterial pattern of the CFFs was imported into COMSOL MultiphysicsThe present study’s overall findings emphasise the capability of the introduced methodology to generate an accurate numerical 2D representation of CFFs, enabling the simulation of a fluid flow through the filters. However, the observed differences in interstitial velocity and permeability highlight the need for further refinement and optimisation of the geometrical pattern to improve the representativeness of the model.Struts during the cutting process, thus preventing their fragmentation and preserving the samples’ integrity.Preparing reproducible CFF samples poses significant challenges, demanding a more accurate and reliable approach. An advanced method involving impregnating CFF samples into castable metallographic resins prior to sectioning will be employed to support and reinforce the CFF The effect of differing meshing methods will also be explored to optimise the sample geometry to better suit the requirements of the numerical 2D macro-structural CFF simulation model. This will, in turn, contribute to securing the best possible sample configuration for accurate analysis and performance evaluation of CFFs.


Musculocontractural Ehlers–Danlos syndrome, caused by biallelic loss-of-function variants for dermatan sulfate epimerase (mcEDS-DSE), is a rare connective tissue disorder. Eight patients with mcEDS-DSE have been described with ocular complications, including blue sclera, strabismus, high refractive errors, and elevated intraocular pressure. However, a case with rhegmatogenous retinal detachment (RRD) has not been reported. We report our findings in a 24-year-old woman who was diagnosed with mcEDS-DSE in childhood and presented to our clinic with an RRD in the left eye. The RRD extended to the macula and was associated with an atrophic hole. The patient underwent scleral buckling surgery and cryopexy with drainage of subretinal fluid through a sclerotomy under local anesthesia. The sclera did not appear blue but was very thin at the sclerotomy site. The patient developed frequent bradycardia during the surgery. Subretinal or choroidal hemorrhages were not observed intraoperatively; however, a peripapillary hemorrhage was observed one day after operation. The retina was reattached postoperatively, and the peripapillary hemorrhage was absorbed after one month. The peripapillary retinal hemorrhages, thin sclera, and bradycardia were most likely due to the fragility of the eye. The genetic diagnosis of mcEDS-DSE played an important role before and during the surgery by alerting the surgeons to possible surgical complications due to the thin sclera. Recent advances in genetic diagnosis have increased the number of cases in which a genetic diagnosis is possible, even for rare diseases. It is expected that the ability to predict the phenotype will identify possible complications that might arise during surgery.CHST14) or dermatan sulfate epimerase  . The. The9]. In earlier studies of RDs in EDS patients, vitrectomy was used to treat RD because of the presence of scleral atrophy and thinning ,14. In aOur study has several limitations. First, this was a single case study, and statistical analysis could not be performed. Nevertheless, scleral buckling surgery was performed successfully. Second, the eye was highly myopic, which is a risk factor for developing RRD and one of the eye complications in mcEDS. Thus, further studies on a larger number of mcEDS-DSE patients are needed.We conclude that cases of mcEDS-DSE with RRD can be successfully treated by scleral buckling surgery. We also conclude that knowing the genetic diagnosis will provide helpful information on possible complications during the intra- and postoperative periods. We recommend that a preoperative genetic diagnosis be made in all cases of inherited connective tissue disorders and that the surgical procedures be designed appropriately.


Syphilis rates in the United Stated (U.S.) have increased steadily for over a decade. Despite over 75 years as the drug of choice for syphilis treatment, controversy persists on optimal duration of therapy with benzathine penicillin G (BPG) for persons with early syphilis, particularly for persons co-infected with HIV. Given the ongoing national shortages of BPG, optimizing duration of therapy is an urgent concern.> 4-fold decline in RPR titer measured at 6 months. Intention to treat (ITT) and per protocol analyses were performed and were similar. ITT analyses are presented here.We conducted a multicenter RCT comparing a single intramuscular (IM) injection of BPG, 2.4 million units to BPG administered for three successive weeks for treatment of early syphilis in persons with and without HIV. The primary outcome of the study was a > 4-fold decline in RPR titer) at 6 months was 76%  0.68-0.82) in the single dose group and did not differ significantly from the three-dose group . There were no treatment failures (> 4-fold increase in RPR titer). Among persons with and without HIV there was no significant difference in RPR response at 6 months: 76% in the single-dose group vs. 71% in the 3-dose group (95% CI 0.05-0.17). Most participants experienced mild to moderate local injection site pain and tenderness.A total of 249 persons with early syphilis were enrolled at 10 participating sites. Most (97%) participants were were categorized as male sex, black race (62%), and 153 (64%) were living with HIV. The syphilis stage distribution was 19% primary, 47% secondary, and 33% early latent and did not differ significantly by HIV status. Serologic response (Treatment of persons with early syphilis with more than a single dose of 2.4 million units of BPG offers no therapeutic benefit irrespective of HIV infection status and was associated with increased rates of injection site discomfort.Edward W. Hook, III, FIDSA, GARPD (Global Antibiotic Resistance Program: Advisor/Consultant|Talis Biomedical: Advisor/Consultant|VISBY Diagnostics: Advisor/Consultant Candice J. McNeil, MD, MPH, BARDA/GSK: Grant/Research Support|Becton Dickinson: Grant/Research Support|Cepheid: Grant/Research Support|Gilead: Grant/Research Support|Hologic: Grant/Research Support|Lupin: study product Julia C. Dombrowski, MD, MPH, Hologic: Grant/Research Support|Mayne Pharmaceuticals: Grant/Research Support


The optimal strategy for fluid management during the first few days of ICU in sepsis patients remains controversial. We aimed to investigate the impact of cumulative fluid balance during the first three days of ICU on the mortality of patients with sepsis.This study analyzed prospectively collected data from the Korean Sepsis Alliance Database, which registered 11,981 sepsis patients from 20 hospitals. We selected three propensity score-matched cohorts consisting of patients with a negative or positive cumulative fluid balance during the first three ICU days: from ICU admission to the first midnight as the D1 cohort, until the second midnight as the D2 cohort, and until the third midnight as the D3 cohort. The propensity score for fluid balance was calculated using covariates including the amount of fluid output during the first three ICU days. The primary outcome was mortality at day 28 in the ICU.P = 0.354) between the two groups. The positive fluid groups in both the D2  and D3  cohorts had significantly higher mortality rates than the negative fluid groups.From a total of 11,981 patients, 2516 patients were included for propensity score matching. After matching in a 1:1 ratio, there were 483, 373, and 392 matched pairs of patients assigned to the D1, D2, and D3 cohorts, respectively. In the D1 cohort, there were no significant differences in mortality at day 28 (hazard ratio [HR], 1.17; 95% confidence interval [CI] 0.85–1.60; In patients with sepsis, a positive fluid balance on the first ICU day was not associated with mortality at day 28. In contrast, cumulative positive fluid balances on the second and third ICU days were associated with higher mortality at day 28.The online version contains supplementary material available at 10.1186/s13613-023-01178-x. The time-dependent bundle has recently become a key strategy for the treatment of sepsis , 2. SincThe influence of a positive fluid balance after the initial fluid resuscitation on survival has been evaluated in several prior studies. A previous pan-European study reported that a positive fluid balance within the first 72 h was the strongest prognostic indicator of death in patients with intensive care unit (ICU)-acquired sepsis . FurtherWe analyzed prospectively collected data of 11,981 patients with sepsis from 20 tertiary referral or university-affiliated hospitals in South Korea between September 2019 and December 2021 as part of an ongoing nationwide, multicenter observational cohort (the Korean Sepsis Alliance registry). The protocols for patient enrollment and data collection have been described previously . All conI = the amount of fluid input at ICU day n, O = the amount of fluid output at ICU Day n, n = ICU Day).In this study, each “time window” was defined as follows: “time zero” referred to the time a patient first entered the emergency room (for those patients admitted to ICU through the emergency room) or the time of screening by the rapid response team (for those patients admitted to the ICU from a ward). The “time before ICU admission” referred to the interval from the time immediately prior to before ICU admission. We defined D1 as the time from ICU admission to the first midnight, D2 as the time from ICU admission to the midnight of the second ICU day, and D3 as the time from ICU admission to the midnight of the third ICU day  score, the presence of septic shock, referring facility, comorbidity, site of infection, adjunct interventions, microbiologic pathogen, and the amount of fluid output . CovariaPatients were divided into a negative or positive fluid balance group in the three matched D1, D2, and D3 cohorts. We compared the mortality rates at 28 days post ICU admission between the negative and positive fluid balance groups in each cohort. Secondary outcomes included the proportions of patients with freedom of mechanical ventilation or that had been discharged from the ICU at 28 days.P values < 0.05 were considered to indicate significance. All analyses were performed using R software version 4.1.2 (R Core Team).Data are presented as numbers with percentages for categorical variables and means plus standard deviations or medians with interquartile ranges (IQR) for continuous variables. A linear mixed model was used to compare continuous variables with the matched cohorts. For time-to-event analysis, the Kaplan–Meier method was used to estimate survival curves and cumulative incidence curves were generated for both the time to discharge alive from the ICU and the time to freedom from invasive ventilation. Time-to-event analysis was right-censored at 28 days. The primary outcome of death within 28 days of ICU admission between the two groups was compared using clustered Cox proportional hazards regression analysis. The results are presented as a hazard ratio (HR) with a 95% confidence interval (CI). The proportional hazard assumption was assessed through an inspection of Schoenfeld residuals. Secondary outcomes were evaluated via competing-risks regression based on a clustered Fine and Gray’s proportional subhazards model. Death before day 28 was considered to be the competing event. This analysis provided sub-hazard ratios (sHRs) and 95% CIs. Two-sided A total of 11,981 patients with a diagnosis of sepsis were admitted to the 20 participating hospitals during the study period  in the D1 model, 0.844  in the D2 model, and 0.836  in the D3 model. The distributions of the SMDs for matched variables revealed that almost all of the patients were closely matched, as indicated by an improvement in the balance after matching  between the two groups, whereas the fluid input was higher in the positive fluid balance group . The net fluid amount remained consistently negative during the first three days in the negative fluid balance group. In the D3 cohort, the fluid output during the first three days was similar between the two groups, while patients in the negative fluid balance group had a lower fluid intake compared with those in the positive fluid balance group, leading to a continuous negative net fluid amount.In the entire unmatched cohort, the cumulative fluid balance during the first three days in the ICU was 1927.8 mL  between the two groups  and D3  cohorts. In addition, there was a stepwise increase in the death hazard at day 28 with increasing cumulative fluid balance in both D2 and D3 cohorts  and ICU discharge at day 28  between the two groups in the D1 cohort. The positive fluid cases in both the D2  and D3  cohorts were less likely to be discharged from the ICU than the patients in the negative fluid group. Additionally, freedom from mechanical ventilation  was consistently lower among patients in the positive fluid balance group in both the D2 cohort  and the D3 cohort .In the D1 cohort, there was no significant difference in mortality at day 28 . Taken together, our results show that cumulative positive fluid balances on the second and third ICU days were associated with increased mortality in patients with sepsis.Numerous studies have been conducted in an attempt to develop an optimal fluid management strategy in the treatment of sepsis that will enhance survival outcomes. Given that higher fluid volumes have been associated with harm in a number of studies of sepsis cases, a prior meta-analysis was conducted of 11 randomized trials in critically ill patients that evaluated the benefits of conservative or deresuscitative strategies  and reported no significant effects on mortality between these two approaches . AnotherOur current findings are also pertinent to the current debates about the timing of the fluid balance in sepsis , 24, 31.The mechanisms of positive fluid balance related to adverse outcomes in sepsis patients remain unclear. However, a conceptual model recommended previously may be of some help in explaining this . In the There were several advantages to our current study over prior investigations. First, we examined the respective effects of the daily fluid balance on each of the first 3 days of ICU care, which was not done in previous studies. Second, our propensity-based analysis of a large prospective database involved well-balanced covariates, thereby allowing for the most reliable comparison of fluid balance effects. For example, comorbidities such as chronic kidney disease, which may have influenced clinical outcomes, were balanced between the two groups. Third, whereas prior studies analyzed the effect of net fluid balance without considering the profile of fluid output, we matched cohorts with similar fluid output profiles to minimize the confounding effect of fluid output on net fluid balance. Fourth, although further double-blinded, interventional, large, randomized controlled trials are needed to confirm these findings, this multicenter nationwide study has a low selection bias. Finally, we also assessed secondary outcomes including freedom of mechanical ventilation and discharge from the ICU, which we found to be consistent with the 28-day mortality results.Nevertheless, our current study should also be interpreted in the context of several limitations. First, we may have missed the possible influence of unmeasured factors despite adjustments for confounders. For example, information about the type of fluid or nutritional support was not available. Second, the fluid profiles of our study subjects were collected by day, so we could not analyze the effects of fluid balance over a shorter time frame during the first three days of ICU care. Moreover, the actual duration of D1 might have differed from patient to patient. Third, no data regarding attempts to reduce fluid accumulation were available. For instance, the use of diuretics was not recorded in the database, and the effect of diuretics on prognosis could not be evaluated in this study. Finally, caution is needed in interpreting the results because this study was conducted on a single ethnic group.In patients with sepsis, a positive fluid balance on the first ICU day was not associated with mortality; however, a positive fluid balance that continued onto the second or third ICU day was significantly associated with mortality. Particularly, the fluid balance on the second ICU day was associated with the highest risk for mortality in these patients.Additional file 1: Figure S1. Study subject flow chart. ICU, intensive care unit; PS, propensity score. Figure S2. Absolute standardized mean differences between negative fluid balance group and positive fluid balance group before and after propensity score matching by the fluid balance of (a) the Day 1, (B) Day 2, and (C) Day 3. The horizontal axis represents the standardized mean differences and red line indicates the absolute standardized mean difference of 0.1. Open dots reflect values prior to matching, and black dots after matching. Matching succeeded in reducing the standardized mean difference within an absolute value of 0.1. SOFA, sequential organ failure assessment; CRRT, continuous renal replacement therapy; ICU, intensive care unit. Table S1. Baseline Characteristics in the full patients of cohorts with sepsis by the first Day 1-3 of fluid balance. Table S2. The profile of fluid input and output in the propensity score matching cohorts by the Day 1-3 of fluid balance.


Clinical implementation of whole genome sequencing (WGS) has been identified as the optimal approach to identify transmission of multidrug-resistant (MDR) organisms in the healthcare setting. Although WGS capabilities are becoming more common in clinical laboratories, analysis and interpretation of results is still a barrier to implementation. The purpose of this study was to evaluate cloud-based automated analytic pipelines to aid with WGS analysis of MDR gram-negative organisms as part of outbreak investigations.Enterobacterales (n: 142), Acinetobacter baumannii complex (n: 14) and Pseudomonas aeruginosa (n:29) isolates were WGS on Illumina sequencing platforms. The FASTQ files were uploaded to AREScloud  and to EPISEQ CS (bioMérieux) for whole genome or core genome multi-locus sequence typing (wgMLST/cgMLST) with dendrogram creation. Results were compared to traditional 7 gene MLST schema. Ease-of-use and additional tools were compared between pipelines.A convenience set of MDR AREScloud and EPISEQ CS generated similar dendrograms for all organisms evaluated . EPISEQ CS provided preset similarity thresholds based on percent relatedness to define clusters simplifying outbreak analysis, whereas AREScloud allowed users to define allelic difference cutoffs. Although the wgMLST/cgMLST clusters aligned with traditional MLST types, not all isolates assigned to a similar MLST were identified as part of a related cluster demonstrating increased resolution of wgMLST and cgMLST phylogenetic assessments. Table 1 compares the features between the two pipelines.Dendrogram generated for Escherichia coli (EPISEQ CS)Dendrogram generated for Escherichia coli (AREScloud)Comparison of features between EPISEQ CS and AREScloud for phylogenetic analysisBoth cloud-based tools perform comparably for assessing phylogenetic relatedness compared to traditional MLST. Preset similarity thresholds available with EPISEQ CS provide ease-of-use for cluster assessment. Additional features, such as resistome and virulome analysis, are available by both pipelines. Predictive WGS-AST is an additional asset available with AREScloud.Karen C. Carroll, MD, Abbott Diagnostics: Board Member|Co-Diagnostics, Inc.: Board Member|Great Basin: Grant/Research Support|Meridian Diagnostics: Grant/Research Support|Pattern Diagnostics: Advisor/Consultant|Pattern Diagnostics: Stocks/Bonds|Qiagen, Inc.: Grant/Research Support|Scanogen: Advisor/Consultant|Scanogen: Grant/Research Support Patricia J. Simner, PhD, Affinity Biosensors: Grant/Research Support|BD Diagnostics: Advisor/Consultant|BD Diagnostics: Grant/Research Support|Entasis: Advisor/Consultant|GeneCapture: Stocks/Bonds|Merck: Advisor/Consultant|OpGen Inc: Board Member|OpGen Inc: Grant/Research Support|OpGen Inc: Honoraria|Qiagen Sciences Inc: Advisor/Consultant|Qiagen Sciences Inc: Grant/Research Support|Shionogi Inc: Advisor/Consultant|T2 Biosystems: Grant/Research Support


The emergence of next‐generation sequencing (NGS) technology has enabled the large‐scale identification of personalised genetic characteristics of colorectal cancer (CRC).n = 535),n = 941)n = 3470).Initially, we identified the correlation between the genomic mutation profile called by MuTect2 and the corresponding tumour purity from three public datasets: The Cancer Genome Atlas (TCGA) . The detailed clinicopathological parameters of the patient cohort are shown in Table .53 Mb. TFIGURE 2Study design and clinicopathological and genomic characteristics of paired‐sample colorectal cancer (CRC) cohort. (A) Study design to evaluate tumour purity factors affecting sequencing results and minimise interference from intra‐tumoural heterogeneity. (B) Heatmap illustrating clinical and genomic data for the 30 paired‐sample CRCs. (C) The distribution of variants under changes in tumour purity. (D) Mutation numbers in routine‐sampling and corresponding precise‐sampling groups with different tumour purities. (E) Common mutation variant allele frequencies (VAFs) before and after precise sampling in relation to tumour purity. (F) Numbers and copy number increments of CNVs before and after precise sampling. (G) Effect of tumour purity on tumour mutational burden (TMB) estimates in precise and routine samples with different tumour purities.The number of mutations after precise sampling was significantly increased in low tumour purity group Figure . The VAFF‐score  and routine‐sampling private mutations as false‐positive mutations (FPMs). The proportion of patients with FNMs gradually reduced as tumour purity increased, while the proportion of patients with FPMs was relatively small and showed no correlation with tumour purity Figure . We evalF‐score . The accF‐score . We furtF‐score . The varF‐score . In contF‐score .Using case 20 as an example, we assessed the impact of tumour cell fraction. on mutation detection with whole exome sequencing (WES). The tumour purity of routine samples evaluated by pathologist and WES were 22.5% and 25%, respectively. The tumour purity was 100% after precise sampling. When compared to the routine sample, the precise sample had more genes with single nucleotide variants (SNVs) and indels Figure . The numIn conclusion, we unveil that tumour purity acts as an independent and significant influencing factor and should be taken into consideration when evaluating genomic characterisation using NGS detection in CRC. Above 30% of tumour purity might be suitable for clinical applications in precision oncology and a higher tumour fraction could promote the accuracy of WES for assessing mutational and clonal landscapes.The authors declare they have no conflicts of interest.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Environmental fungi are threats to personal and public health. Fungal in vitro diagnostics help diagnose invasive fungal infections (IFIs), but clinicians remain underinformed about their use and interpretation. Given the increasing use of social media to share infectious diseases–related content, we designed and implemented a multisite Twitter-based curriculum focused on IFIs and related diagnostics.Questions were posted through a dedicated Twitter account twice weekly over 8 weeks. We surveyed clinicians at 3 US academic centers before and after completion of the curriculum and interviewed a subset of participants. We undertook quantitative and qualitative evaluations and reviewed Twitter analytics.P = .005) in the percentage of correct answers in the pre- versus postcurriculum knowledge assessments. Perceived benefits included a well-executed curriculum that facilitated engagement with appropriately detailed tweetorials from a dedicated Twitter account. Perceived barriers included lack of awareness of tweetorial posts and timing, competing priorities, and the coronavirus disease 2019 pandemic. The Twitter account accrued 1400 followers from 65 countries during the 8-week period. Tweets with multiple-choice questions had a median of 14 904 impressions , 798 engagements , and an engagement rate of 6.1% .We surveyed 450 participants. One hundred twenty-one participants (27%) completed the knowledge assessment precurriculum, 68 (15%) postcurriculum, and 53 (12%) pre- and postcurriculum. We found a significant increase (72% vs 80%, Educators can leverage social media to share content with a large audience and improve knowledge while being mindful of the barriers associated with implementing a curriculum on social media. We implemented a multisite Twitter-based curriculum focused on fungal infections and diagnostics. We demonstrated a wide dissemination of content and observed a significant increase in knowledge. This article highlights ways in which educators can leverage social media to improve knowledge. The clinical utility of in vitro diagnostics varies according to factors that impact analytic performance  and predictive value . The serum Platelia GM enzyme immunoassay is cleared as an aid to diagnose invasive aspergillosis, with best performance in people with high risks associated with neutropenia ) of respondents felt that having knowledge of IFI risk factors and test characteristics of BDG and GM was either important or very important to their clinical practice. Comparing the pre- to postvignette survey responses, participants reported significant increases in confidence in their ability to name IFI risk factors, indications for serum BDG, indications for serum GM, and reasons for a false-positive serum BDG P < .05; . We foun = .005) . Knowled = .005) .In the postvignette survey, 10% (7/68) of respondents stated that they completed >50% of the Twitter clinical vignette–based questions. Eighty-two percent were satisfied or very satisfied with the quality of the Twitter questions, and 69% considered the Twitter questions to be difficult. Ninety percent believed that the content was clear, and 100% felt that the content was informative. Finally, 65% thought that Twitter was effective as a learning tool, and 65% would participate in a Twitter-based curriculum again.During the 8-week period, the @FilaMentor account accrued 1400 followers from 65 countries. The majority were from the United States (US) (53%), followed by India (7.9%), the United Kingdom (5.1%), Mexico (3.7%), Saudi Arabia (3.3%), and Canada (3.1%) . Within The perceived benefits of the curriculum included program characteristics that filled knowledge gaps and facilitated engagement . SpecifiThere were several perceived barriers to participation in the curriculum . One barWe herein describe a multisite Twitter-based curriculum focused on fungal infections and diagnostics. Most notably, despite limited promotion of the curriculum outside of the intended participants, there was widespread dissemination of content both nationally and internationally. Comparing the pre- to postvignette survey responses, we found a significant increase in knowledge assessment scores. Participants enjoyed the content and reported that they would participate in a Twitter curriculum again. Finally, participants noted several barriers to participation that warrant reflection when designing and evaluating Twitter-based medical education curricula.Few data have been published regarding the implementation of ID-related social media curricula. In a single-center study, investigators used both Twitter and Facebook to design an antimicrobial stewardship curriculum focused on creating awareness of resources and improving knowledge. Questions were posted on both platforms followed by published manuscripts or links to internal resources. Among IM resident participants, pre- to postcurriculum knowledge assessment test scores increased significantly (from 60% to 65%) . In our Rates of engagement with our questions and tweetorials were high, with a median engagement rate of 6.1%. While there are no published data regarding mean or median rates of engagement with medical education content on social media, according to the 2023 Social Media Industry Benchmark Report, the median engagement rate across all industries is 0.037% [Although participants felt our curriculum was well-done and facilitated engagement, we encountered substantial barriers to implementing and studying our curriculum during the coronavirus disease 2019 (COVID-19) pandemic. At all 3 sites, IM residents reported feeling inundated with weekly surveys. Concern for survey fatigue among graduate medical education (GME) trainees is not a new problem. In a qualitative study of 33 GME trainees, negative emotions such as annoyance and survey fatigue were identified as barriers to completing surveys. The trainees noted that a high volume of survey invitations and emails played a role in their low response rates . Other sConsistent with data from other reports, the topic of burnout during COVID-19 was mentioned several times in the interviews. Among 300 residents who responded to a survey about stress and burnout during the COVID-19 pandemic, 27% reported high stress, 30% reported anxiety and work overload, and >50% reported symptoms of burnout . While eGiven the limited experience and published guidance, we wish to provide guidance to educators who aim to implement longitudinal curricula on social media. First, support from key stakeholders to allot dedicated time to learners to complete questions and read tweetorials will help ensure the curriculum's success. To help facilitate this, educators may wish to carve out the beginning portion of time set aside for education  to allow trainees to complete the question and tweetorial. This need not happen every day for a given curriculum. For example, when we asked participants how often they prefer to receive Twitter questions and tweetorials, the most common response was once a week. Second, to ensure that trainees are aware of the question and tweetorial posts, educators may wish to create a unique Twitter account dedicated exclusively to the educational content, and they may wish to inform learners how to follow and turn on tweet notifications for that specific account. Third, implementing a virtual curriculum with limited social interaction is challenging. Interviewees highlighted the value of in-person education and program promotion. Even outside of a pandemic, if feasible, to improve participation and engagement in a longitudinal Twitter-based curriculum, educators may wish to dedicate a portion of a highly attended conference to introducing the curriculum and teaching residents how to use Twitter and set up relevant notifications. Educators may also wish to meet trainees on the wards or in work rooms to promote the curriculum rather than advertising it exclusively virtually. These measures may help participants feel more connected as part of a collective learning experience to optimize learner engagement. For educators who wish to explore Twitter content with learners or address misinformation/disinformation on social media, we suggest relevant review articles , 31.This study has some limitations. One limitation is the low response rate and potential for selection bias. Second, because of our low response rate, we were unable to meaningfully stratify our results by PGY level, site, or provider level. Third, although our observed improvement in knowledge assessment scores was statistically significant, an increase from 72% to 80% may not be educationally significant. Fourth, Twitter does not facilitate analysis of question responses at an individual level, which prevented us from conducting a correlation analysis between the performance on Twitter questions and the Qualtrics survey questions. Fifth, although the multisite program is a strength, there was variability in the environment and culture at each site with respect to social media presence, timing of additional surveys, and rates of SARS-CoV-2 infections. Sixth, this study measured the knowledge impact of the curriculum at 3 US institutions and may not reflect its impact at other US or international sites. Finally, although Twitter is a rapidly evolving platform, we feel that our approach may be used for similar emerging platforms. Two additional possible future avenues of study include assessing the durability of knowledge retention from a social media curriculum and incorporating measurement of impact on clinical practice.In this program, we implemented a multisite Twitter-based curriculum focused on fungal infections and diagnostics. The strengths of this project include its multisite design, mixed-methods evaluation, use of a novel Twitter handle to permit assessment of reach of the curriculum, and its international reach. We demonstrated a wide dissemination of content over social media and observed absolute increases in knowledge across all 4 learning objectives, with significant increases in 2 of these. Our data highlight ways in which educators can leverage social media to share content with a large audience to improve knowledge, especially in a time where virtual learning is here to stay.ofad594_Supplementary_DataClick here for additional data file.


Monitoring the physiological changes of organelles is essential for understanding the local biological information of cells and for improving the diagnosis and therapy of diseases. Currently, fluorescent probes are considered as the most powerful tools for imaging and have been widely applied in biomedical fields. However, the expected targeting effects of these probes are often inconsistent with the real experiments. The design of fluorescent probes mainly depends on the empirical knowledge of researchers, which was inhibited by limited chemical space and low efficiency. Herein, we proposed a novel multilevel framework for the prediction of organelle-targeted fluorescent probes by employing advanced artificial intelligence algorithms. In this way, not only the targeting mechanism could be interpreted beyond intuitions but also a quick evaluation method could be established for the rational design. Furthermore, the targeting and imaging powers of the optimized and synthesized probes based on this methodology were verified by quantitative calculation and experiments. Subcellular organelles, as subunits of cells, play an indispensable role in different physiological processes. The imbalance of biologically active species in them will cause their dysfunction, which has a serious impact on the health of organisms ,2. FluorIn recent years, with the explosive growth of biomedical data and the rapid development of computer software and hardware, artificial intelligence has increasingly penetrated deep into various aspects of drug research and development and has greatly boosted the process –22. EspeHere, a new method integrated with advanced artificial intelligence with quantitative calculation was proposed to help provide new insights into the accurate design of specific subcellular organelle probes . Firstly, we collected the currently published fluorescent probes targeting subcellular organelles and chemical compounds with known subcellular localization. Then, we established a pretreated molecular library. On this basis, we developed a multilevel framework that consisted of a series of prediction models by employing advanced-artificial intelligence methods. The first level was a classification model (B-PvsC model) to uncover the reason why the subcellular organelle targeting of fluorescent probes were different from commonly seen chemical compounds. The second level was a binary classification model (B-MvsP model) expected to focus on mitochondrial targeting. The third level was a multiclassification model (M-PvsP model) expected to predict specific subcellular organelles targeting of a probe further. The last level was a colocalization model (B-McoL model) to estimate the targeting effect of a probe. Afterward, by taking advantage of the detailed explanations and summarized rules of these models, we designed several groups of probes on the basis of different fluorescence mechanisms and estimate them by our framework. Then, on this basis, we selected the fluorescent probes with better performance to synthesize them and verified their accuracy and biological application by experimental methods. In addition, we further combined the means of quantum chemistry to perform quantitative calculations on the synthesized molecules to explain their spectroscopic properties. With the above inspiring strategies, a method that enabled more accurate prediction of organelle-targeted fluorescent probes was successfully constructed. Thus, it would help researchers to design and construct novel organelle-targeted fluorescent probes more accurately and rationally, especially for mitochondria-targeted ones, and allow more effective detection of specific bioactive molecules, thereby advancing the field.A total of 1,661 organelle-targeted fluorescent probes and 614 compounds that were not fluorescent probes (Np-Compounds) were manually collected from >10 thousand publications over the recent 10 years (2012–2022) Table . These dIn addition, for mitochondria-targeted fluorescent probes, we have also attempted to estimate the colocalization effect by predicting their correlation coefficients (B-McoL model). For the 1,005 mitochondrial targeting probes, their colocalization in the measurement system by different colocalization dyes was collected. The 914 data, for which the dyes were MitoTracker Green and MitoTracker Red (MTR), and numerical correlation coefficients were available, were selected and deduplicated according to InChIKey. Then, the arithmetic mean of multiple records for the same molecule was adopted. Since the colocalization data would only be reported when they obtained an expected probe with a good colocalization ability, the correlation coefficients were usually high. Specifically, the correlation coefficients of the retained 896 unique probes were centered overwhelmingly between 0.8 and 1. Therefore, we took 0.8 as the threshold and labeled 811 probes with correlation coefficients not <0.8 as the positive and 85 probes <0.8 as the negative. In addition, a synthetic minority oversampling technique  was adopThe optimal B-PvsC model for detecting mitochondria-targeted fluorescent probes and Np-Compounds was explored with a wide range of descriptors and algorithms. The best performance was achieved by ECFP4-LR model with an accuracy (ACC) of 0.93 and 0.95 for the cross-validation (CV) and test set, respectively . The areThe B-MvsP model tried to distinguish the probes targeting mitochondria and other organelles with mitochondria as positive and the other 5 organelles as negative. By comparing molecular descriptors/fingerprints and algorithms, the best model is the combination of MACCS fingerprints and LightGBM, with accuracy of 0.858 and 0.843 for the CV and test set, respectively (Table). The AUC of 0.916 ± 0.01 (Mean ± SD) for CV showed good performance Fig. . The 2D The M-PvsP model was designed to further specify the more detailed localization of fluorescent probes among the 6 kinds of organelles. By comparing the predictive power of different kinds of descriptors and algorithms, the best model was the MACCS-LightGBM. The accuracy was 0.783 and 0.809 for the CV and test set, and the AUC was 0.904 and 0.932 . The AUC for CV was quite high reaching 0.98 Fig. . For theTo explore the relationships between molecular structures and the organelle-targeting effects, the model explanation was performed and rules were summarized. When focused on mitochondria-targeted ones, the B-MvsP model using MACCS fingerprints and LightGBM algorithm showed the best predictive power and good robustness. Therefore, we made an explanation for the model based on the interpretable SHAP (SHapley Additive exPlanations) values and the feature importance generated by LightGBM. Because of the differences between the selected algorithms, the feature importance calculated will not be identical, but the most important features will always have a higher score. Among the 166 MACCS keys, more than half of the Top50, Top30, and Top15 most important features selected preferentially via both methods overlapped, and 88% of the Top50 were mainly associated with heteroatoms such as nitrogen, oxygen, and phosphorus. A visualization of the 9 most important MACCS keys codetermined in the Top15 of SHAP and LightGBM methods is shown in Fig. To validate the practicality of the explanation and rules, and evaluate the predictive power of the framework, we first designed a series of fluorescent molecules based on excited-state intramolecular proton transfer (ESIPT), twisted intramolecular charge transfer (TICT), and ICT, which were 3 representative fluorescent mechanisms. Then, among these fluorescent molecules with different mechanisms, we further distinguished them into mitochondrial dyes and probes that could be specifically used for ROS, enzymes, and viscosity imaging in mitochondria according to their structural properties, which were 3 important biomedical applications of mitochondria-targeted probes. According to our plan, firstly, we designed 451 molecules of ICT and 70 molecules of ESIPT, respectively. Then, some of these molecules were connected with the corresponding ROS response sites ,32 and aThe molecular library mentioned above was fed into our multilevel framework to predict their organelle-targeting and colocalization effects. The predicted results are shown in Fig. To help us select the optimized probes, in addition to the rules summarized above, the physicochemical properties, synthetic accessibility, and safety were evaluated by an ADMETlab platform  for compWe first optimized the ground-state structure of our selected probes and then calculated the ultraviolet (UV) absorption of each compound based on different functionals based on this structure. As shown in Table The detailed synthesis process and schematic diagram can be found in the Supplementary Materials.After obtaining P1-ALP, we first measured the UV spectrum and fluorescence spectrum of P1-ALP. As shown in Fig. After obtaining P2-VIS, we measured the UV and absorption of P2-VIS. As shown in Fig. S18, the maximum absorption of P2-VIS is 550 nm. Because of the strong TICT effect of P2-VIS, P2-VIS basically has no fluorescence emission. Subsequently, we measured the fluorescence intensity of P2-VIS at different viscosities. As shown in Fig. Similarly, for P3-ROS, we measured the UV and fluorescence spectra of the probe, and the results showed that the maximum absorption of P3-ROS was 425 nm . After P3-ROS reacted with hydrogen peroxide, the UV absorption was 360 nm, and the fluorescence emission was 535 nm . We measured the pH effect, selectivity, and response time respectively . It was shown that the probe enabled a specific response to hydrogen peroxide in the pH range of 6 to 9 within 30 min. According to the response experiment of P3-ROS to hydrogen peroxide at different concentrations, the probe P3-ROS could detect quantitatively hydrogen peroxide between the hydrogen peroxide concentration of 0 to 10 μM . However, in the M-PvsP model, the sample size gap between categories increased obviously, resulting in categories with smaller sample sizes  being more likely to be misclassified to the category with a larger proportion of sample size . This is because the classifier calculates the probability of each sample falling into each class in the prediction and determines which class the sample most likely belongs to by comparing the maximum of the 6 probabilities. Since the ROC curves are class skew independent, but related closely to the probability the classifier assigns to all samples in each class, it is clear that the AUC values for each class in the test set are still high . Thus, the M-PvsP model we constructed did successfully distinguish 6 types of organelle-targeting probes from the others. In this multiclassification model with imbalanced sample distribution, the probability values may reveal its true categories better than the output labels.On the basis of the above statistical calculations, it was indicated that the positive charge was important for mitochondria-targeting molecules, which was in accordance with the mechanistic studies that positive charges could be pumped into mitochondria driven by mitochondrial membrane potential . This exIt was worth noting that the probes targeting the nucleus and lysosome were also positively charged, which means that the specificity of the mitochondria-targeted molecules could not rely only on the explanation based on the positive charge. In terms of molecular structure, the molecules targeting lysosome are highly similar to those targeting mitochondria, which makes it difficult to distinguish them from the view of the structure. Moreover, there were some special fluorescent molecules with multiple targeting abilities, which also affects the ability of predicted molecules to target mitochondria. These phenomena remind us that the probe design based on the consideration of multiple important factors should be better and a more refined multiclassification model may help.Given the abovementioned mechanisms and analysis, it should be pointed out that there is still more that can be done to improve the predicting ability. First, the limited reaction types and structural scaffolds restricted the application domain of the models, which can easily lead to misclassification for the organelle categories with a small amount of data mentioned above. It suggests that we need to enlarge the data size of the model, which is a challenge in itself. Secondly, under the current prediction framework, we can combine the prediction results with structural similarity to make a better decision regarding the confusing organelle-targeted ones. Finally, the development of some new descriptors capable of elucidating the mechanism of fluorescent molecules will help to characterize the structure of such molecules, thereby improving the accuracy and specificity of the models.During the screening of optimized probes, almost all designed molecules were predicted to have good mitochondrial targeting ability, as the molecular design of this library was based on the inspiration and rules obtained from the above artificial intelligence models. However, it was this situation that posed a challenge for us to select more reasonable probes. Therefore, we need to consider not only the targeting ability of these probes but also some important additional properties, as well as the complexity of the physiological environment and the synthesis conditions in the laboratory. For example, here, the logP value of P1-ALP was not prominent in the whole library, but it was still selected after comprehensive consideration. Notably, the colocalization experiments showed a colocalization coefficient (0.85) of P1-ALP lower than that of P2-VIS (0.93) and P3-ROS (0.94), which confirmed the rule we concluded: Lipophilicity was friendly to mitochondrial targeting.According to our experimental results, the compound structures we predicted on the basis of the above descriptors have good accuracy for fluorescent molecules with different fluorescence mechanisms. The results of our synthesized P1-ALP, P2-VIS, and P3-ROS with ESIPT, TICT, and ICT properties show that P1-ALP, P2-VIS, and P3-ROS are not only suitable for detecting respective analytes in vitro and in cell imaging but also can accurately target mitochondria for the imaging of each detected object in the mitochondria.In summary, we proposed a new method that integrated artificial intelligence with quantitative calculation to enable the rational design of organelle-targeted fluorescent probes. We firstly collected high-quality datasets concerning the organelle-targeted molecules and established a multilevel prediction framework by systematically comparing different algorithms and molecular descriptors, from which we obtained the structural features and rules of fluorescent molecules targeting mitochondria. Then, we applied these rules to design a library of mitochondria-targeted fluorescent probes with different fluorescence mechanisms. After assessment based on the framework and important physicochemical properties, 3 optimal probes were selected and synthesized. Their fluorescence mechanisms were then verified by quantitative calculation. As expected, in the experiments, they successfully achieved mitochondria targeting and subsequently detect ALP, viscosity, and hydrogen peroxide, respectively. Therefore, we believe that this work not only provides a great reference value for seeking fluorescent probes related to subcellular imaging but also helps to make a step forward in the intelligent design of molecular probes.In this work, different kinds of molecular descriptors and fingerprints were calculated to represent the physicochemical properties and structural features of molecules. The MOE software  was used to calculate 2D descriptors, which consists of 206 descriptors in continuous and discrete values. The RDKit  and CDK In order to explore the most suitable machine learning models for the designed multilevel framework, 10 classical algorithms that cover tree ensemble models and linear models were employed, including simple decision tree , random A customized feature selection pipeline was adopted to deprecate redundant features and avoid unnecessary computational costs. MOE 2D descriptors with variance of zero were dropped. If high correlation (> 0.95) were found between 2 descriptors, one of them was reserved. An interpretable explanatory method named SHAP  was usedFirstly, we used MOE to perform a conformational search on the ground-state structure of the compounds. After obtaining the structure with the lowest energy, we optimized the ground-state structure of each compound based on PBE0/Def2-SVP, and then because of the different charge transfer properties of each compound, we performed the structural optimization of excited states based on PBE0 and CAM-B3LYP, respectively. After obtaining the most stable structure, we calculated the single-point energies based on PBE0/Def2-TZVP and CAM-B3LYP/Def2-TZVP, respectively, to obtain more accurate excitation energies. In the calculation of the potential energy surface, we use the flexible scanning method to obtain the ESIPT and TICT or PICT potential energy surface.Unless otherwise stated, all reagents and chemicals were purchased from qualified suppliers with required purities. All glassware was dried before use. Nuclear magnetic resonance spectra were measured on a Bruker AVANCE III HD 500-MHz spectrometer with tetramethylsilane as the internal standard. Mass spectrometry was performed on an Agilent Technologies 6530 quadrupole time-of-flight liquid chromatograph-mass spectrometer. UV-vis absorption spectra were performed on a UV-2550 scanning spectrophotometer . Fluorescent spectra were recorded on a Hitachi F-2700 equipped with a 1-cm quartz cell. Dynamic light scattering measurements were performed at 25 °C on Zestier Nano ZS .2 atmosphere. Cells were seeded in petri dishes  for overnight culture. Then, the solution of selected probes (10 μM) and MTR (1 μM) were added to the cells and incubated for 30 min at 37 °C. After washing with phosphate-buffered saline 3 times, cells were imaged with a laser scanning confocal microscope .MCF-7 cells and Hela cells were purchased from the Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37 °C in a 5% CO