The haemostatic status of dogs with canine parvovirus (CPV) enteritis, within 24 h of admission after initial fluid administration, has been described previously, but the haemostatic status at admission and after standard fluid resuscitation, as well as after initial fluid redistribution, has not been investigated previously. The objective of this study was to characterise the haemostatic status at admission and describe the effect of crystalloid fluid resuscitation on haemostatic variables in dogs with CPV enteritis. Twenty-seven client-owned, hospitalised dogs with confirmed natural CPV infection and 15 healthy age-matched controls were included in a prospective, observational clinical study. The volume of resuscitation fluid, haematocrit (HCT), platelet count, thromboelastography (TEG) variables, antithrombin (AT) activity, fibrinogen- and C-reactive protein (CRP) concentrations were measured in all dogs at admission, after fluid resuscitation and, in 10 dogs, after receiving an additional 3 hours of maintenance-rate crystalloid fluids. For the CPV group at admission, the median TEG reaction time (R) and maximum amplitude (MA) or clot strength, as well as the median HCT, fibrinogen and CRP concentrations, were significantly increased compared to the controls. After fluid resuscitation, median R was significantly shorter, MA significantly increased and HCT and AT activity significantly decreased compared to admission values. The haemostatic variables remained unchanged after 3 h of maintenance-rate crystalloid therapy. The increased clot strength present in dogs with CPV enteritis at admission was exacerbated after fluid resuscitation and persisted for hours after large-volume crystalloid fluid administration. Canine parvovirus (CPV) is a small, non-enveloped single-stranded deoxyribonucleic acid (DNA) virus that preferentially infects tissues with rapid cell turnover, including the intestinal epithelium and bone marrow Parrish . IntestiViscoelastic methods of coagulation testing such as thromboelastography (TEG) and thromboelastometry (TEM) combine the evaluation of the traditional plasma components with the cellular components of haemostasis and allow for the evaluation of clot formation, clot strength and fibrinolysis  or clot strength, a TEG variable, increased fibrinogen concentration and decreased antithrombin (AT) activity, has been reported in dogs with CPV enteritis, within 24 hours of admission after initial fluid administration  due to anaemia or haemodilution with crystalloid fluid supplementation has been reported to result in a relative increased clot strength, or MA, while a high HCT generally results in a relatively decreased MA , using kaolin-activated TEG, found no significant difference in R, at 33% dilution, but reported a reduced α-angle at 67% dilution. The MA was significantly decreased at both dilutions , nearly 30% of the infused fluid remains in the intravascular space 30 min after the cessation of fluid therapy  dogs with CPV enteritis would have a higher MA at admission compared to control dogs and (2) that fluid resuscitation would result in a further increase in MA that outlasts the expected fluid redistribution.Client-owned dogs presenting to the Onderstepoort Veterinary Academic Hospital (OVAH) between January 2016 and January 2017 with naturally occurring CPV enteritis, and healthy control dogs, were enrolled in a prospective, observational study. Dogs in both the CPV and control groups were between 8 weeks and 9 months of age and weighed more than 4.5 kg. Control dogs were presented for vaccination, ovariohysterectomy or castration and were deemed healthy based on the absence of vomiting or diarrhoea in the preceding 14 days, having had no contact with dogs affected by CPV enteritis, a normal clinical examination and a peripheral blood smear negative for blood-borne parasites. Dogs presenting with clinical signs associated with CPV enteritis such as lethargy, anorexia, vomiting, diarrhoea, dehydration and/or hypovolaemic shock were tested for CPV. Dogs that tested positive using CPV antigen ELISA , were preliminarily diagnosed with CPV enteritis and were enrolled in the CPV group. Eligible cases were only enrolled if they were admitted for in-hospital treatment and had not received any treatment prior to admission. Infection with CPV was confirmed by faecal electron microscopy. Dogs that received any medication known to interfere with normal haemostasis – such as corticosteroids, non-steroidal anti-inflammatory drugs or anticoagulant dugs, during hospitalisation or one month prior to hospitalisation – and dogs that had previously received a transfusion of any blood products were excluded from both the control and CPV groups. Dogs were deemed to be in hypovolaemic shock if they showed clinical signs indicative of abnormalities in perfusion such as tachycardia (> 140 beats per minute), peripheral vasoconstriction with cool extremities, hypothermia (< 37.5 °C), a prolonged capillary refill time (> 2 s), poor peripheral pulse quality and mental dullness  once daily, 20 mg/kg ampicillin IV every 8 h and 15 mg/kg metronidazole IV every 12 h, during the duration of data collection. Fentanyl constant rate infusion (CRI) was administered at 3 μg/kg/h in dogs with severe abdominal discomfort. Any other required treatment was administered after the last blood sample was collected. The control group did not receive any treatment. None of the dogs received any drugs known to affect haemostasis during the resuscitation period, and 0.9% NaCl without additional heparin was used to flush IV catheters.Clinical parameters were recorded for each dog in the control and CPV groups, and percentage dehydration or presence of hypovolaemic shock was recorded in the CPV group. The first blood collection was performed at admission, prior to any treatment or procedures for the control and CPV groups. Blood was again collected in the CPV group after fluid resuscitation (hypovolaemic dogs) or 90 min – 110 min of fluid therapy (dehydrated dogs). A third sample was collected from 10 randomly selected dogs in the CPV group, by blinded draw of a card (A or B) from an envelope, 3 h after fluid resuscitation while on a 10 mL/kg/h CRI of LRS. Blood was collected via jugular venipuncture, using a 21G needle into evacuated serum and citrate tubes in the recommended order  boluses of 10 mL/kg bodyweight were given over 10 s – 60 s, and repeated every 15 min, until the normalisation of five out of seven clinical variables  was achieved (defined as the completion of fluid resuscitation). In addition, if dehydration was identified in these hypovolaemic dogs, rehydration fluids were initiated at the same time, which entailed the replacement of the estimated percentage dehydration (over 24 h) in addition to maintenance fluid requirements (60 mL/kg/day – 80 mL/kg/day) using LRS. In the hypovolaemic group, blood collection was repeated within 15 min of fluid resuscitation completion, while still on rehydration fluids. For the dehydrated dogs with CPV without signs of hypovolaemia, rehydration entailed the same fluid plan as dehydrated hypovolaemic dogs (excluding the fluid boluses). Repeat blood collection was performed at a pre-determined time of 90 min – 110 min of fluid therapy in all of the dehydrated dogs, while still on rehydration fluids, to be of a similar time period to what was expected for fluid resuscitation in the hypovolaemic dogs. Blood collection was not repeated in the control group.To assess the effect of initial fluid redistribution after the initial fluid resuscitation of 90 min – 110 min of fluid therapy, a blood sample was also collected as previously described, in 10 randomly selected dogs with CPV, 3 h after an additional 10 mL/kg (approximating maintenance-rate fluids and for standardisation across hydration status) of LRS (defined as the CRI group).Blood samples from admission (CPV and control groups) and after fluid resuscitation (dehydrated and hypovolaemic CPV groups) were analysed. Citrated whole blood was kept at room temperature for 30 min before a tissue factor-activated TEG analysis using the TEG 5000 Thrombelastograph Hemostasis Analyzer system  was performed. The principal investigator performed all of the TEG analyses. Machine blood counts  to determine HCT and platelet count were performed on citrated whole blood at the time of each TEG assay. The HCT and platelet counts were corrected for the dilutional effect of the citrate anticoagulant . Hypovolaemic and dehydrated dogs (subgroups) were grouped together as the CPV group. The Shapiro-Wilk test was used to assess the data for normality. The Chi-Square test was used to compare sex proportions and the Mann-Whitney U test was used to determine differences between dehydrated and hypovolaemic dogs after the CRI of LRS infusion. Correlations were determined using Spearman’s rank correlation coefficient for the CPV group at admission and after fluid resuscitation; subgroup analysis was also performed. A p-value of less than 0.05 was considered significant. Data are presented as median and interquartile range (IQR), unless specified otherwise.After fluid resuscitation, the dogs in the CRI group were evaluated as a whole, and subgroup analysis for the dehydrated and hypovolaemic dogs was performed. The Wilcoxon signed-rank test was also used to determine differences between variables from fluid resuscitation until after the CRI of LRS in the 10 randomly selected CPV dogs, and in the dehydrated and hypovolaemic subgroups. The Mann-Whitney The study was reviewed and approved by the University of Pretoria Animal Ethics Committee (AEC number V092-15). Informed written consent was obtained from all owners.Twenty-eight dogs with naturally occurring CPV enteritis and 15 healthy control dogs were enrolled in the study. One dog was excluded after a negative result on faecal electron microscopy. Canine parvovirus-infected dogs consisted of 16 dogs that presented with hypovolaemic shock and 11 with dehydration. All dogs with hypovolaemic shock were also dehydrated and therefore received fluid boluses as well as concurrent rehydration and maintenance rate fluids. The signalment of the CPV and control groups is included in p < 0.001); equivalent to 26.5 mL/kg (19.8 mL – 39.0 mL) and 10 mL/kg (8.1 mL – 11.3 mL), respectively.The principal investigator collected all of the samples. There was no significant difference in the timing of sampling between the dehydrated and hypovolaemic CPV subgroups. The median volume of fluid administered between the first and second blood collection for the hypovolaemic group was 286 mL (246 mL – 470 mL) and was significantly higher compared to the 54 mL (51 mL – 116 mL) for the dehydrated group , the α-angle was significantly smaller (p = 0.037) and the MA significantly larger (p < 0.001) in the CPV group compared to the control group. The hypovolaemic group had significantly longer R and K, and larger MA compared to the control group . The dehydrated group only showed a larger MA compared to the control group (p = 0.018). No significant differences were found between the hypovolaemic and the dehydrated groups at admission.p = 0.028 and p = 0.020, respectively), and the α-angle and MA significantly larger  in the CPV group compared to their values at admission. In the hypovolaemic group, the MA was significantly larger (p = 0.007), while no significant TEG variable changes were seen in the dehydrated group from admission to after fluid resuscitation. No significant differences were found between the hypovolaemic group and the dehydrated group after fluid resuscitation.After fluid resuscitation, the median R and K were significantly shorter  in the CPV group compared to the controls; however, the platelet count was similar between groups. Compared to the controls, the median fibrinogen and CRP concentrations were significantly higher in the CPV group (p < 0.001 for both). The median AT activity was similar between groups. Both the hypovolaemic and dehydrated groups had significantly higher fibrinogen and CRP concentrations than the control group (p < 0.001). No significant differences were found between the hypovolaemic and dehydrated groups at admission.p < 0.001) and the platelet count significantly higher (p = 0.034) compared to their values at admission. The median CRP concentration and AT activity were significantly lower compared to their values at admission , although CRP remained markedly increased above the reference interval. In the hypovolaemic and dehydrated subgroups, both the HCT  and CRP  decreased significantly. Antithrombin activity decreased significantly in the hypovolaemic group (p = 0.007), yet increased significantly in the dehydrated group (p = 0.045). The fibrinogen concentration remained above the assay detection limit. No significant changes were seen in the CRI group, or the hypovolaemic and dehydrated subgroups ; and MA a moderate positive correlation with the platelet count . Within the dehydrated subgroup at admission, AT activity was strongly correlated with the α-angle .For the CPV group at admission, R had a moderate negative correlation with AT activity  in the CPV group. Antithrombin activity had a moderate negative correlation to the change in R . In addition, the volume of fluid administered had a strong positive correlation with the decline in AT activity . The change in CRP had a moderate positive correlation with R  and K  and a moderate negative correlation with α-angle . In the hypovolaemic group, MA retained a strong positive correlation with the platelet count , while the volume of fluids administered had a moderate positive correlation with the decline in AT activity  and a moderate negative correlation with the platelet count . In the dehydrated group, MA retained a strong positive correlation with the platelet count  and the volume of fluid administered had a strong positive correlation to the change in AT activity from admission .Post fluid resuscitation, MA retained a moderate positive correlation with platelet count  and delayed clot initiation  compared to healthy control dogs. The MA increased, clot initiation time shortened and AT activity decreased after crystalloid fluid resuscitation in dogs that received large volumes of fluids. The increased MA and decreased AT activity persisted up to 3 h after resuscitation with crystalloid fluid therapy, while still receiving a constant rate of fluids.The R and K variables were significantly longer and the α-angle significantly smaller for the CPV group at admission, indicating delayed clot initiation and amplification compared to the healthy control group. The prolonged R and K were also present in the hypovolaemic subgroup at admission compared to the controls, but not in the dehydrated subgroup. The R, K and α-angle variables measure the time to, and rate of, clot generation  and increased clot strength (larger MA) after fluid resuscitation compared to admission may be as a result of the crystalloid fluid therapy, the progression of disease or a combination of these two factors. In the CPV group, the magnitude of decrease in HCT after fluid resuscitation correlated moderately with the volume of fluid administered, as would be expected. The significant decrease in HCT after fluid therapy may have contributed to the increased MA, as reported in an in vitro dilution rate of 33 mL/kg in the study by Morris and co-workers that reported the prolongation of K, reduced α-angle and decreased MA  from admission blood sampling to the initiation of fluid therapy, the speed at which fluid boluses were given and the time between fluid boluses, although all of these were case-appropriate. The objective of the study was to determine haemostatic changes after fluid resuscitation, and the true effect of the minor time differences could not be determined from the study. The lack of a CPV group that did not receive fluids did not allow for determination of the cause of the increase in the hypercoagulable state after fluid administration, but would not have been ethically acceptable. The measurement of the D-dimer concentration and fibrin degradation products were not performed in this study. No measurable D-dimer concentrations were found in a previous study evaluating TEG in CPV  and further increased clot strength, as evidenced by larger MA in dogs with CPV that were hypovolaemic at presentation. Increased clot strength persisted for hours after large-volume crystalloid fluid administration.


Primary endpoint was progression-free survival (PFS). Analyses of PFS by baseline factors, including liver tumour burden, ALP elevation, and target lesion size, were performed using Kaplan-Meier estimates; hazard ratios (HRs) with corresponding 95% CIs were estimated using Cox regression.177Lu-Dotatate versus octreotide LAR 60 mg in patients with low (< 25%), moderate (25–50%), and high (> 50%) liver tumour burden , and normal or elevated ALP , and in the presence or absence of a large target lesion . Within the 177Lu-Dotatate arm, no significant difference in PFS was observed amongst patients with low/moderate/high liver tumour burden (P = 0.7225) or with normal/elevated baseline ALP (P = 0.3532), but absence of a large target lesion was associated with improved PFS (P = 0.0222). Grade 3 and 4 liver function abnormalities were rare and did not appear to be associated with high baseline liver tumour burden.Significantly prolonged median PFS occurred with 177Lu-Dotatate demonstrated significant prolongation in PFS versus high-dose octreotide LAR in patients with advanced, progressive midgut NET, regardless of baseline liver tumour burden, elevated ALP, or the presence of a large target lesion.Clinicaltrials.gov: NCT01578239, EudraCT: 2011-005049-11The online version of this article (10.1007/s00259-020-04709-x) contains supplementary material, which is available to authorized users. P = 0.017) [The liver is the dominant site of metastatic disease amongst patients with stage IV well-differentiated neuroendocrine tumours (NET) . High li= 0.017) .177Lu) is a beta- and gamma-emitting radionuclide [90Y), 177Lu has lower maximum and mean beta particle energies and maximum and mean soft-tissue penetration depths of 1.7 and 0.23 mm, respectively [177Lu effectiveness has not been evaluated in a randomised controlled trial.Tumour size is often considered a prognostic factor for patients treated with radiolabelled somatostatin analogue (SSA) . Lutetiuonuclide . Compareectively , consideectively , 17, 18.177Lu-Dotatate efficacy and toxicity, we conducted a post hoc analysis of the NETTER-1 trial, the only prospective phase 3 study of a radiolabelled SSA [177Lu-Dotatate every 8 weeks for four cycles, or high-dose octreotide LAR 60 mg every 4 weeks. At the time of primary endpoint data analysis (24 July 2015), median PFS was not reached (NR) in the 177Lu-Dotatate arm and was 8.4 months in the control arm  [177Lu-Dotatate in the clinically relevant domains of global health status, physical functioning, role functioning, diarrhoea, pain, and fatigue [To assess the impact of these potential prognostic and predictive factors on lled SSA . In NETT13–0.33) . Health- 24 July 15, media177Lu-Dotatate treatment efficacy outcomes (PFS), TTD in QOL, and hepatic toxicity rates. We evaluated the predictive and prognostic power of elevated ALP, whether presence of ≥ 1 target lesion >3 cm in diameter impacted PFS benefit with 177Lu-Dotatate, and whether baseline tumour size correlated inversely with tumour shrinkage rates.We assessed the impact of baseline liver tumour burden on Eligible patients were aged ≥ 18 years with locally advanced or metastatic, low-, or intermediate-grade (Ki-67 ≤ 20%) NET originating in the midgut with radiologic disease progression  while receiving a standard dose of octreotide. All target lesions were required to be somatostatin-receptor-positive. Hepatic exclusion criteria were total bilirubin > 3× ULN and serum albumin ≤ 3.0 g/dL, unless prothrombin time was within normal range.177Lu-Dotatate (administered every 8 weeks) along with intramuscular (IM) octreotide LAR 30 mg every 8 weeks (followed by maintenance octreotide LAR 30 mg every 4 weeks) or to high-dose octreotide LAR 60 mg every 4 weeks. Patients were stratified by highest tumour uptake on somatostatin receptor scintigraphy and by duration of prior treatment with constant-dose octreotide LAR (≤ 6 or > 6 months).Patients were randomised to four cycles of The trial protocol was approved by the institutional review board or independent ethics committee at each institution. The trial was performed in accordance with the principles of the Declaration of Helsinki, International Conference on Harmonisation Good Clinical Practice guidelines, and all applicable regulations. All patients provided written informed consent.Baseline liver tumour burden was estimated by blinded central radiology review  and categorised into subgroups of low < 25%), moderate (25–50%), or high (> 50%) tumour burden according to liver tumour volume divided by total liver volume by computed tomography (CT) or magnetic resonance imaging (MRI). The thresholds chosen were similar to those described in prior phase 3 studies evaluating SSAs in NETs 5%, moder.P-values were estimated using a Cox regression model with randomised treatment, liver tumour burden at baseline and liver tumour burden × randomised treatment interaction term as covariates. The primary data analysis cutoff was 24 July 2015.PFS curves for each treatment arm and median PFS with corresponding 95% CIs were generated using Kaplan-Meier estimates, stratified by liver tumour burden, and the log-rank test was used for within–treatment arm comparisons of PFS. HRs with corresponding 95% CIs and P-values were generated using the methodology described above.PFS curves were generated for each treatment arm, stratified by baseline ALP , and the log-rank test was used for within–treatment arm PFS comparisons. HRs with corresponding 95% CIs and P-values were generated using the methodology described above.Patients were stratified into two subgroups based on the presence or absence of at least one target lesion >30 mm in diameter at any body site on CT or MRI at baseline. This approximate size threshold has been described in previous literature as distinguishing ‘large’ tumours from smaller ones in animal studies of peptide receptor radionuclide therapy (PRRT) , 22. PFSA mixed model repeated measures (MMRM) analysis included study visit, baseline tumour size (≤ 30 mm and > 30 mm), and baseline tumour size × study visit interaction as fixed effects, and was used to evaluate the effect of baseline tumour size on least squares mean percentage change in tumour size from baseline to week 72 .Assessment of grade 3 or 4 liver function test (LFT) abnormalities  was stratified by tumour burden categories described above. The analysis comprised all patients who underwent randomisation and received at least one dose of trial treatment . Adverse events in NETTER-1 were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.02.TTD of QOL  was defined as the time from randomisation to first deterioration ≥ 10 points  compared with baseline on EORTC QLQ-C30 and GI-NET21. TTD was estimated using Kaplan-Meier methodology and stratified by liver tumour burden subgroup: low (< 25%) or moderate to high (≥ 25%).177Lu-Dotatate patients, 114 high-dose octreotide patients) were enrolled in NETTER-1; 223 received at least one dose of study drug and were eligible for safety analysis . Supplementary TableIn total, 231 patients  in those with low burden; NR versus 8.7 months in those with moderate burden ; and NR versus 5.4 months in those with high burden .Statistically and clinically significant prolongation of PFS with 177Lu-Dotatate arm, no significant difference in PFS was observed with low, moderate, or high baseline tumour burden (log-rank P = 0.7225). However, within the high-dose octreotide arm, there was a significant correlation between liver tumour burden and PFS, with median PFS of 9.1, 8.7, and 5.4 months for low, moderate, and high burdens, respectively (log-rank P = 0.0169).Within the 177Lu-Dotatate was observed amongst patients with normal and elevated baseline ALP, with nearly identical HRs for progression or death in both prognostic groups  in the normal ALP group and NR versus 5.8 months  in the elevated baseline ALP group.In each treatment arm, 112 patients had evaluable baseline ALP. Statistically and clinically significant prolongation of PFS with 177Lu-Dotatate (log-rank P = 0.3532) or high-dose octreotide arm (log-rank P = 0.0911).No significant difference in PFS was observed amongst patients with normal versus elevated ALP in the 177Lu-Dotatate arm, 128 large tumours (>30 mm diameter) were identified, of which 89 (70%) were liver tumours; in the high-dose octreotide arm, 134 large tumours were identified; 93 (69%) were liver tumours. Regardless of presence or absence of a large baseline lesion, median PFS was significantly prolonged amongst patients treated with 177Lu-Dotatate versus high-dose octreotide . However, there was also clinically and statistically significant benefit of 177Lu-Dotatate amongst patients with ≥ 1 large target tumour; median PFS was NR in the 177Lu-Dotatate arm versus 8.5 months in the high-dose octreotide arm .Amongst target lesions in patients within the P = 0.3566). However, absence of a large target lesion was associated with improved PFS in the 177Lu-Dotatate arm (log-rank P = 0.0222), although median PFS was NR in both groups.The presence or absence of a large baseline lesion did not impact the PFS of patients receiving high-dose octreotide  regardless of baseline size. At 72 weeks, least squares mean shrinkage was 29% and 14% in the ≤ 30 mm and > 30 mm groups, respectively. There was a significant interaction of baseline tumour size by time of visit (P = 0.0085) within the 177Lu-Dotatate-treated group, indicating that liver tumour size shrinkage over time differs by baseline size.To assess whether baseline liver tumour size correlates with radiographic tumour shrinkage in patients receiving ory Fig. . Tumour 177Lu-Dotatate arm versus 6.1 months in the high-dose octreotide arm . In patients with moderate/high tumour burden (≥ 25%), the median TTD of global health status was NR in the 177Lu-Dotatate versus 6.0 months in the high-dose octreotide arm . The median TTD of other clinically relevant QOL domains stratified by tumour burden are shown in Supplementary TableIn patients with low tumour burden (< 25%), median TTD of global health status was 28.8 months in the Grade 3 and 4 LFT abnormalities were rare and did not appear to be associated with high baseline liver tumour burden in either arm Table . Because177Lu-Dotatate has not been well established, partly owing to lack of randomised studies, which are often necessary to identify predictive factors. Two retrospective studies of 177Lu-Dotatate have demonstrated that tumour burden ≥ 25% is associated with a shorter median OS in multivariate analyses ; however, the relationship with PFS was not investigated [177Lu-Dotatate versus high-dose octreotide. Indeed, the HR for PFS benefit in the high tumour burden group was nearly identical to the benefit in the low burden cohort. When evaluating each treatment arm separately, high tumour burden was a negative prognostic factor for PFS in the high-dose octreotide arm but did not correlate with negative outcomes in the 177Lu-Dotatate arm, suggesting that 177Lu-Dotatate may mitigate the negative impact of tumour burden.The impact of liver tumour burden and largest tumour size on outcomes with stigated . Our ana177Lu-Dotatate versus high-dose octreotide in the high ALP group was nearly identical to the benefit in the normal ALP group. A study of patients treated with 177Lu-Dotatate has demonstrated ALP elevation (> 120 IU/L) to be a negative prognostic factor in terms of OS, but did not assess PFS [Similar findings were observed with ALP elevation as with tumour burden, which is consistent with the association of ALP with tumour burden . The HR sess PFS .177Lu-Dotatate versus high-dose octreotide, with a 94% improvement in risk of progression or death (HR 0.06). PFS benefit with 177Lu-Dotatate versus high-dose octreotide was also seen with at least one large target lesion (HR 0.21). However, in those receiving 177Lu-Dotatate, absence of a large target lesion was associated with improved PFS. Mean tumour shrinkage with 177Lu-Dotatate correlated with baseline tumour size, being highest in target lesions ≤ 30 mm. These outcomes indicate the effectiveness of 177Lu-Dotatate across a spectrum of tumour sizes but also suggest that its effectiveness is particularly high in smaller tumours. Randomized trials are necessary to prove or disprove the hypothesis that longer-range radionuclides  should be used in combination or as an alternative to 177Lu-based PRRT in patients with large tumours.In this study, presence or absence of a large (> 30 mm) target lesion did not impact the PFS of patients receiving high-dose octreotide . This suggests that the effect of octreotide is independent of tumour size. Patients lacking a large target lesion had a particularly pronounced PFS benefit with 177Lu-Dotatate has a clinically relevant beneficial impact on overall QOL as well as on specific NET-related symptoms regardless of tumour burden. However, when stratified by tumour burden, most QOL results were not significant owing to the small number of patients in each cohort (data not shown).The QOL findings suggest that 177Lu-Dotatate in patients with high tumour burden owing to the potential for radiation hepatitis. Data from NETTER-1 did not validate this hypothesis. LFT elevations were rare and did not appear to correlate with baseline tumour burden. It is important to note, however, that safety findings in patients with tumour burden > 50% do not necessarily imply that treatment is equally safe in patients with extreme tumour burden . A limitation of this study is that central readers did not specify the patients with extreme tumour burden (> 90%), and therefore no specific safety analysis in that subgroup was possible.Concerns exist regarding the safety of 177Lu-Dotatate demonstrated significant prolongation in PFS versus high-dose octreotide in patients with advanced, progressive midgut NET, regardless of baseline liver tumour burden, elevated ALP, or presence of a large target lesion. 177Lu-Dotatate is effective across a spectrum of tumour sizes, but its effectiveness is particularly high in smaller tumours, potentially supporting early treatment in patients with progressive disease. Clinically relevant LFT abnormalities were rare and were not associated with high baseline liver tumour burden.In summary, ESM 1(DOCX 15 kb)ESM 2(DOCX 15 kb)ESM 3(DOCX 16 kb)ESM 4(DOCX 282 kb)ESM 5(DOCX 16 kb)


We propose to use Twitter data as social-spatial sensors. This study deals with the question whether research papers on certain diseases are perceived by people in regions (worldwide) that are especially concerned by these diseases. Since (some) Twitter data contain location information, it is possible to spatially map the activity of Twitter users referring to certain papers . The resulting maps reveal whether heavy activity on Twitter is correlated with large numbers of people having certain diseases. In this study, we focus on tuberculosis, human immunodeficiency virus (HIV), and malaria, since the World Health Organization ranks these diseases as the top three causes of death worldwide by a single infectious agent. The results of the social-spatial Twitter maps  reveal the usefulness of the proposed sensor approach. One receives an impression of how research papers on the diseases have been perceived by people in regions that are especially concerned by these diseases. Our study demonstrates a promising approach for using Twitter data for research evaluation purposes beyond simple counting of tweets. In total, we downloaded 17,295 PubMedIDs from papers regarding HIV, 26,595 PubMedIDs from papers regarding tuberculosis, and 13,974 PubMedIDs from papers regarding malaria.Altmetric.com via the PubMedID. 8,442 of the HIV-related papers (48.8%) were tweeted by in total 55,506 tweets. 11,139 of the tuberculosis-related papers (41.9%) were tweeted by in total 85,737 tweets. 8,403 of the malaria-related papers (60.1%) were tweeted by in total 73,111 tweets. Overall, the papers regarding these three diseases have been mentioned in tweets more often than medical papers in general [see The PubMedIDs were imported into our in-house PostgreSQL database and matched with the Twitter data from eral see .Altmetric.com database. The three sets of tweets were downloaded via the Twitter API using R [The Tweet ID was exported from the  using R  with the using R  and RCur using R  and stor using R . Functio using R  were use using R , 56. Frohttps://grid.ac/) for obtaining a whitelist of existing cities and countries. Only that location information was kept which contained a city name and a country name from the GRID database. Usage of the GRID database introduces a potential bias towards city names with research institutes and English city and country names. For the countries, we added “USA” and “UK” (requiring a comma or whitespace before “UK”). In both data sets, location information and GRID data, we removed non-standard characters  by only keeping the standard alphabet letters, whitespaces, and some punctuation characters . In addition, both data sets were converted into lower case characters for matching.One problem with the free-text user location information is that some users seem to become very imaginative. In order to reduce wrong location information, we needed to filter the location information for meaningful entries. We imported the city and country names from the Global Research Identifier Database . The remaining location strings were passed to the Google API via the R package ggmap  if the lion data  shared bThe R package tidyverse  was usedhttps://en.wikipedia.org/wiki/Censorship_of_Twitter).It should be considered in the interpretation of the Twitter data that censorship of Twitter in certain countries exists which “refers to Internet censorship by governments that block access to Twitter, or censorship by Twitter itself”  in health research. One important outcome of these and similar studies was “the surveillance of influenza outbreaks with comparable accuracy to traditional methodologies” . The resOn a related note, Sakaki et al.  coined tAltmetric.com. The company classifies users who tweet about papers into four groups: researcher, practitioner, science communicator, and member of the public [see also Altmetric.com explains the groups as follows:In order to receive information on the people tweeting on research about tuberculosis, malaria, and HIV, we used classifications of user profiles provided by see also . Altmetr“Member of the public–somebody who doesn’t link to scholarly literature and doesn’t otherwise fit any of the categories belowResearcher–somebody who is familiar with the literaturePractitioner–a clinician, or researcher who is working in clinical sciencehttps://help.altmetric.com/support/solutions/articles/6000060978-how-are-twitter-demographics).Science communicator–somebody who links frequently to scientific articles from a variety of different journals / publishers” . Furthermore, we can use it without restrictions to profiles with geographical location information.The use of Altmetric.com categorizations for the characterization of Twitter users in this study is associated with two problems: (1) the analyses cannot be restricted to only those Twitter users for whom we have geographical location information. (2) Altmetric.com defines the group ‘members of the public’ as people who do not link to scholarly literature. However, our dataset is based on only tweets including links to papers. Toupin, Millerand, and Larivière  proposedThe classification scheme by Toupin et al.  is more Faculty and students: accounts whose general interest lies in higher education or the realm of research;Communicators and journalists: accounts whose general interest lies in the transmission of information at a higher scale ;Professionals: accounts who may have an interest in engaging with research papers according to their job ;Political: accounts who have political interests in engaging with papers ;Personal: accounts that self-describe themselves using personal interests Institutions and organizations: accounts whose interests represent a group of people;Bots: accounts that describe themselves using keywords related to automated activity;Journals and publishers: accounts that represent journals or scientific publishers.Toupin  has provAltmetric.com is used. There seems to be a large proportion of members of the public in the dataset of this study. Using the tweets of the profiles as social sensors, we investigate in the following sections whether research on a certain disease  reaches the people that are especially concerned by the disease (regions with many people having the disease). Using the location information converted into geo-coordinates (see section 3), we mapped Twitter activity on certain papers . Each tweet is represented by a single dot on the map.The results in One of the problems with Twitter data in the context of this study is that Twitter activity is generally high where much research is done . The results are reported in The relevant information in The results of the Poisson regression analysis are shown in Twitter data cannot only be used as social sensors on the country level, but can be restricted to a single country. We would like to demonstrate the approach based on US HIV-related data. We calculated Poisson regression models with number of HIV cases and number of papers as independent variables and number of tweets as dependent variable. The use of altmetrics and especially Twitter data in research evaluation (impact measurements) has been assessed very critically in the past years , 74. TheIn this study, we propose to abstain from using Twitter data as simple counts, but to use additional meta-data, which are accessible for single tweets and Twitter users. For Pershad et al. , “througScience mapping has become an important method in research evaluation . The litThe results of the social-spatial Twitter maps and regression models reveal that the combination of both methods is useful to answer our research question. We received an impression of how research papers on tuberculosis, malaria, and HIV have been perceived by people in regions which are especially concerned by these diseases. The maps give a spatial overview and a first impression; the regression models quantify the attention research papers have received regionally. For example, the comparison of the regression model results for tuberculosis, malaria, and HIV reveal that research papers might be of specific public interest with respect to HIV. The percentage in expected counts is higher than those for malaria and tuberculosis. The HIV Twitter analysis that focused on US states shows that the relationship between public attention and number of HIV cases is higher than the relationship between public attention and research activity. The results might suggest how important social media platforms are in diffusing research into areas where diseases are more prevalent but research outputs are low. The methods proposed in this study might be a good supplement to previously introduced methods investigating whether research efforts (public funds) measured in terms of publications respond to local  burdens of diseases .Our study might demonstrate an interesting approach for using Twitter data for research evaluation purposes. The data should be used, however, with care. A first important point concerns the location information from Twitter. Wouters et al.  identifiA third point refers to the content of tweets: can we assume that tweets deal with the “right” papers which are helpful with respect to certain diseases? Other papers might be more helpful. Furthermore, we cannot assume in every case that “true stories” on papers are distributed on Twitter. The results by Vosoughi, Roy, and Aral  show thaA fourth point refers to dependencies in the Twitter data. Single tweets might have mentioned more than one paper, and one Twitter user might have posted more than one tweet on diseases. These dependencies might distort the empirical results. In this study, we did not consider these dependencies in the data structure, since we analyzed the data on aggregated levels. Future studies could try to analyze the data on a lower level to control the results for these dependencies.We recommend that our proposal of using Twitter data should be tested by other research groups (active in scientometrics). According to Thelwall , “indicaIn this study, we focus on the health care sector to demonstrate our proposal of using Twitter data as social-spatial sensors. Our proposal, however, is not only restricted to this sector. Sakaki et al.  list som


Malignant tumours are one of the major diseases that seriously endanger human health. The characteristics of their invasion and metastasis are one of the main causes of death in cancer patients, and these features cannot be separated from the participation of various molecules‐related cells living in the tumour microenvironment and specific structures. Tumour invasion can approximately be divided into several specific steps according to the movement of tumour cells. In each step, there are different actions in the tumour microenvironment that mediate the interactions among substances. Researchers are attempting to clarify every mechanism of the tumour dissemination. However, there is still a long way to the final determination. Here, we review these interactions in tumour invasion and metastasis at the structural, molecular and cellular levels. We also discuss the ongoing studies and the promise of targeting metastasis in tumour therapy. Primary tumours can be treated by surgical resection, but cancer mortality increases significantly once the tumour metastasizes. Tumour metastasis, as an important signal of cancer staging, has become a hot topic in cancer treatment. Therefore, research on tumour invasion and metastasis is particularly important. The metastasis caused by carcinomas is formed following the completion of a complex succession of cell‐biological events, collectively termed the invasion‐metastasis cascade.The tumour microenvironment, where tumour cells live, includes a variety of cells (such as cancer‐associated fibroblasts (CAFs), tumour‐associated macrophages (TAMs), cancer stem cells (CSCs) and endothelial cells) and extracellular matrix proteins that are predominant in tumour metastasis invasion.2At present, the TNM staging system is the most widely used staging system in the world.3As mentioned previously, there are several steps in the invasion‐metastasis cascade: local invasion through the surrounding extracellular matrix (ECM) and stromal cell layers, intravasation into the lamina of blood vessels, surviving the rigours of transport through the vasculature, arresting at distant organ sites, extravasation, surviving the foreign microenvironments to form micro‐metastasis and finally, reviving the proliferative programmes at metastatic sites, thereby generating macroscopic and clinically detectable neoplastic growths.3.1The so‐called local invasion is that the cancer cells located in the primary tumour enter the surrounding matrix and then migrate into the adjacent normal tissue parenchyma,Matrix metalloproteinase (MMPs) and epithelial‐mesenchymal transition (EMT) mediate this process. Cells expressing MMPs release growth factors, which are involved in the degradation of the basement membrane as well as other extracellular matrix (ECM) components and promote the proliferation of cancer cells.3.2Lymphatic and blood diffusion are two ways of infiltration, and the latter seems to be the main mechanism of metastasis.After cancer cells invade the surroundings and intravasate, these cells, named CTCs (circulating tumour cells), are located between the primary tumour and the site of transmission and shoulder the task of surviving under various pressures to reach distant organs. In the absence of integrin‐dependent adhesion to the extracellular matrix component, epithelial cells usually initiate anoikis. Anoikis, a form of apoptosis, is a special programmed cell death induced by the loss of contact between cells and the extracellular matrix.3.3Although CTCs can theoretically spread to a variety of secondary sites, clinicians have noticed that a single cancer type forms metastatic organ in a limited number of target organs Figure C. There After the tumour cells are captured, the next step is to infiltrate into the substance of the distant tissue. Although most models of metastasis include an extravasation step early in the process, study shows that metastasis is mainly induced by the proliferation of tumour cells attached to the vascular endothelium while the extravasation of tumour cells is rare.In addition, as discussed earlier, the neovascularization of primary tumours is zigzag and leaky. However, microvessels in normal tissues at a distance are normal‐like, resulting in reduced permeability. For example, disseminated cancer cells that attempt to reach the brain's parenchyma must cross the blood‐brain barrier.3.4The microenvironment of the metastatic site usually differs from that of the primary site, which means that cancer cells, at least in the beginning, are not fully adapted to their newly discovered home. The diversity may include the types of stromal cells, the components of the extracellular matrix, the effective cytokines or even the microstructure of the tissue itself Figure D. Some rIf only disseminated cancer cells are first exposed to the microenvironment of foreign tissues and successfully survive, they still do not guarantee proliferation. Conversely, most disseminated tumour cells appear to have experienced slow depletion for several weeks or even months, otherwise to remain dormant as microcolonies for long periods of time.4Some kinds of endothelial adhesion molecules, such as integrin and selectin, can regulate the infiltration of T cells in the tumour microenvironment as long as desmosomes and tight junctions are present. However, little is known about the genetic mechanism of these molecules when they affect immune cells in cancer patients.+ T cell, and the promotion of the immune escape of tumour cells. Furthermore, the activation of this kinase can affect the adhesion of plaques by affecting the activity of patchy enzymes.The expression of the Ras gene can be used as a G protein to participate in the signal transduction process of tumour cells.In addition to the related genes, cell adhesion molecules participate in the signal transduction of tumour cells. Many cell adhesion molecules have been discovered with a wide spectrum of responsibilities, including recruiting, activating, elongating and maintaining.4.1The classic cadherin is a multifunctional cell adhesion receptor. It is a multidomain, trans‐membrane protein in which the extracellular domain forms the homotypic, adhesive interaction while the intracellular domain interacts with the actin cytoskeleton through the catenin family of adaptor proteins.4.2The integrin family is a heterodimer cell surface receptor family that perceives these changes and triggers a series of cellular responses by forming physical connections inside and outside the cell, thus allowing bidirectional ‘integration’ signals to control cell adhesion, migration, proliferation, survival and differentiation. At present, eighteen α subunits and eight β subunits have been reported, which can form at least twenty‐four heterodimer receptors.During local invasion, integrins can regulate multiple signals and affect the transmission of tumours in primary sites. When tumour cells escape into blood vessels or lymphatic vessels, they must adapt to survive without ECM adhesion. At this point, integrin controls the central role of growth and survival without relying on anchorage, as integrin β‐1 promotes prostatic anchorage‐independent growth,4.3Selectin is a surface lectin that regulates the adhesion of leucocytes, platelets and endothelial cells in the bloodstream, including L‐selectin, E‐selectin and P‐selectin. These proteins are expressed in platelets and vascular endothelial cells. Among them, L‐selectin plays a good role in leucocyte recruitment, mediating inflammation and lymphocyte homing.4.4The formation of neovascularization can satisfy the substance supply of the tumour. Neovascularization is an essential condition for tumour growth and plays an important role in the process of tumour invasion and metastasis. Tumour cells host epithelial cells, endothelial cells and other cells and can secrete a variety of active factors to induce tumour angiogenesis. For example, growth factors secreted by tumour cells can stimulate this process, so these growth factors are called angiogenic factors, including heparin‐binding growth factors or fibroblast growth factor families, transforming growth factor α, vascular osmotic growth factor (VPF) and vascular endothelial growth factor (VEGF). These growth factors promote the proliferation of vascular endothelial cells and induce angiogenesis by combining with corresponding target cells.Epithelial to mesenchymal transition (EMT) is the highlight affecting changes in adhesion factors and related signalling pathways and promotes cell migration during tumour metastasis.4.5Proteolytic enzymes are required in several critical stages of tumour progression, such as the hydrolysis of ECM, which requires a series of specific proteolytic enzymes to complete the process. These proteolytic enzymes include MMPs, matrix‐degrading enzymes and cysteine proteases. For example, MMPs catalyse various proteins, including collagen, and matrix degradation, resulting in the remodelling of the structure of the ECM, making it more conducive to tumour cell migration and thereby promoting tumour metastasis. The major MMPs associated with tumour angiogenesis are MMP‐2, MMP‐9 and MMP‐14, followed by MMP‐1 and MMP‐7. MMP‐3, MMP‐7, MMP‐9 or MMP‐16 directly dissects the matrix‐bound vascular endothelial growth factor, leading to a change in the bioavailability of the modified VEGF molecule. MMPs can also cut the extracellular domain of cadherins in endothelial cells and destroy the intercellular junctions. Multiple angiogenesis promoters can induce the expression of MMPs in endothelial cells, and MMPs, in turn, enhance the biological activity of angiogenic factors. MMP substrates also include adhesion molecules and apoptosis‐mediated factors that regulate cell adhesion and change the ability of tumour cells to metastasize.5TAMs and CTCs play a catalytic role in both the local invasion of tumour cells into the vascular cavity and the survival of tumour cells under blood flow conditions. Tumour‐derived CSF‐1 can recruit macrophages to home and produce a large number of associated cytokines, such as EGF, VEGF, TGF‐β, to enhance cell invasiveness CTCs are cells similar to primary tumour cells and are usually used as markers of distant metastasis. The most important characteristic of CTCs is that they undergo EMT reversible transformation Figure B and theCSCs are defined as cancer cells with the stem‐cell‐like ability. The original CSCs may participate in metastasis or a new type of CSC derived from the first one or another cell in the tumour that acquires metastatic traits could do so. The formation of such a ‘metastatic CSC’ might be observed as EMT.+ tumour cells. The upregulation of the S1pr1 gene can activate STAT3 and promote tumour growth and metastasis. Some studies have shown that S1PR1 leads to the sustained activation of STAT3 and that STAT3 can induce the expression of the S1pr1 gene. Both activities promote the expression of STAT3 in tumour cells and play an important role in tumour progression.When tumour cells prepare to intravasate into the parenchyma of distant tissue, myeloid‐derived suppressor cells (MDSCs) may facilitate this process. These cells mainly help to form a prosurvival environment for metastasis and prepare for the subsequent colonization and reproliferation of a large number of tumour cells. Under the action of many tumour‐derived factors, medullary tumour cells proliferate and mobilize into the blood to act on distal target organs. MDSCs in the pre‐metastatic microenvironment can promote further amplification through the S1PR1‐STATS signalling pathway and help tumour cells penetrate into the circulatory system Figure C. S1PR1 + T cells and CD8+ T cells by immune actions.Dendritic cells (DCs) are heterogeneous and specialized antigen‐presenting cells (APCs) that have an unequalled capacity to initiate primary immune responses. Growing evidence suggests multiple cross‐talks between DCs, and other cells from tumour microenvironment can contribute to antitumour immunity or immunological tolerance by regulating T cell differentiation and responses + GPR77+ CAFs promote tumour formation and chemoresistance by providing a survival niche for CSCs.Cancer‐associated fibroblasts (CAFs) are important components of the tumour microenvironment and appear in almost every stage of invasion. The toxic tumour microenvironment may induce the phenotypic differentiation of multiple resident precursors into CAFs.In general, all the various cells mentioned above enhance the ability of tumour cells to metastasize and act at different stages of invasion by directly secreting proteins or indirectly make contributions with the help of other cells. In addition, the microfilament microtubules and mesenchyme in the cytoskeleton are also associated with the tumour, but to what extent can these factors influence the cancer progression and the detailed mechanisms are still unclear, which calls for further study.6The current treatments for tumour limited mainly in surgery, chemotherapies and radiotherapies. The interdisciplinary collaboration among nanotechnology, biology, chemistry, pharmacology, oncology and other disciplines has led great changes in these three primary therapy.Targeted drugs are imperative in the treatment of cancer. As molecules or their preparations endowed with targeting ability, they form a relatively high concentration of drugs in the target area, so as to improve the efficacy and inhibit the toxic and side effects to normal tissues and cells.Aside from things above, latest methods for cancer treatment are emerging in an endless steam. For example, Chitosan‐modified PLGA polymeric nanoparticle system kills cancer cells and reduces pain by facilitated to apoptosis, especially for metastatic cancers.The field of cancer immunotherapy has recently received a significant attention. The treatment of targeted tumour metastasis prevents or slows down tumour metastasis, which prolongs the life of tumour patients and improves the clinical survival rate. However, it is still difficult for some of the targeted drugs to focus on tumour tissues and then affect other healthy tissues, resulting in adverse reactions due to the lack of specificity. More research is needed on the safety, specificity and stability of targeted therapy for tumour metastasis. In addition, the combined targeted delivery of treatments is an important method to enhance the therapeutic efficiency and reduce adverse side effects for current therapies.7In this paper, we discussed the mechanisms of promoting tumour metastasis from the genetic, molecular, cellular and structural levels according to the time sequence of tumour metastasis and invasion. It is clear that the mechanisms are not independent, and the specific substances of each time course have many similarities. The molecules or cells that interact with each other among different pathways that are cross points, forming a network to coordinate tumour metastasis. Tumour metastasis is a major cause of tumour death. Controlling tumour metastasis can prolong the survival time and improve the quality of life of cancer patients. Therefore, studies of tumour metastasis can provide a variety of ideas for cancer treatment. Since there have already been a number of targeted tumour therapy drugs or treatment methods, anticancer drugs with more targeted, higher safety trends are developing. However, all studies have shown that homologous molecules may have expression in different tissues, that is, their expression in tumour organs is not special enough to be safe targets, and the origin point is still not appearing. Drugs manage to not only kill cancer cells but also destroy normal tissues and reduce the normal functioning of the body simultaneously. Furthermore, the same molecule may present the opposite functional characteristics at different stages of tumour development and this tendency usually hard to estimate, pulling one hair and affecting the whole body. When we are looking at an old tree, we are still doing research on its broomy branches and looking forward to digging into its main root. Generally, the research of tumour metastasis is developing continuously, but it is still not enough to support antitumour therapy. We hope that the study of tumour metastasis will not only broaden the number of molecules but also deepen the research on each related substance and determine the ultimate key point of tumour metastasis.The authors declare that they have no conflicts of interest to this work.All authors wrote the manuscript and ZF designed the figures; JL and XZ provided guidance and revised this manuscript. All authors approved the final manuscript.


To report a case of Optic Disc Pit (ODP) maculopathy exacerbated during pregnancy.A 30-year-old female developed unilateral blurry vision at 10-weeks gestation. Ophthalmic examination revealed left eye reduced visual acuity (VA) with the presence of subretinal fluid temporal to the disc extending to the fovea. On Spectral Domain Optical Coherence Tomography (SD-OCT) subretinal, and intraretinal fluid was confirmed. Laser photocoagulation was tried in an attempt to prevent surgical intervention without success; subsequently, pars plana vitrectomy, internal limiting membrane peel and gas tamponade was performed. Three-weeks later, a full thickness macular hole developed, and repeat surgery was performed. Nine-months after the second surgery the macular hole was closed with near complete resorption of edema.No trigger factors for ODP maculopathy have been reported before. We report a case of worsening ODP maculopathy during pregnancy with good visual outcome after surgical intervention. Optic disc pit (ODP) is a rare congenital defect of the optic nerve characterized by a grayish oval depression at the optic disc, most frequently found at the inferotemporal region –8. Even A 30-year-old female was initially referred to our clinic with a four-day history of central blurry vision in the left eye.Her past medical history was significant for dysautonomia treated with oral fludricortisone and propranolol, and HEELP syndrome that developed during her previous pregnancy. Past ocular history was only positive for mild myopia (− 1.50 D) in both eyes. She had been followed yearly by a general ophthalmologist prior to consultation. Family history was non-contributory.Her exam revealed a best corrected visual acuity (BCVA) of 20/25 in the right eye and 20/30 in the left eye. Intraocular pressure (IOP) was 16 mm of Hg in both eyes. Her external ocular exam, anterior segment and vitreous were normal bilaterally. Retinal exam in the right eye was unremarkable, and in the left eye an optic nerve pit was noted with the presence of intraretinal fluid temporal to the disc, sparing the fovea.  and subretinal (SR) fluid. Fig. a Due to Maculopathy is a well described condition occurring in 25–75% of patients with ODP and being responsible for significant vision loss in up to 65% of the patients , 4, 9–11The exact mechanism of maculopathy development is still unknown; the vitreous, cerebrospinal fluid, leakage from blood vessels and fluid from choroid through Bruch’s membrane are four possible origins that have been postulated , 14, 15.There is no universally accepted management, but the current standard treatment of ODP maculopathy is PPV, with posterior vitreous detachment induction, ILM peeling, peripapillary laser, fluid-air exchange and gas tamponade , 11, 13.Laser photocoagulation at the temporal disc margins was introduced as a conservative treatment to prevent further fluid accumulation by creating a barrier between the ODP and subretinal space , 7, 11. FTMH has been associated with chronic macular detachment, leading to irreversible visual loss , 11. ManTo date, there are no known triggers for developing ODP maculopathy. There is one case report which describes childbirth as a trigger to developing ODP maculopathy due to differences between intraocular pressure and intracranial pressure, but nevertheless our patient presented with worsening ODP maculopathy during pregnancy. The relationship between pregnancy and childbirth and ODP maculopathy progression is unknown but might be related to hormones or Valsalva phenomenon present during this period of time. Moreover, poor visual prognosis is a common finding in these patients especially after FTMH formation. We present a case with a good visual outcome after surgical intervention under local anesthesia during pregnancy. Despite a severely edematous macula and thinned fovea leading to a full thickness macular hole after the initial surgery, a second surgery proved successful in closure of the macular hole and substantial visual improvement. Surgery performed under local anesthesia with consent from the attending obstetrician and maternal fetal medicine specialist may be a successful route to stabilize, improve, and spare vision loss in this susceptible population.


Metastasis is facilitated by the formation of pre-metastatic niches through the remodelling of the extracellular matrix (ECM) promoted by haematopoietic and stromal cells. The impact of these primed sites is pronounced for intraperitoneal metastases, where the cavity-exposed ECM supports the attachment of the disseminating tumour cells. Likewise, implantation of biomaterial scaffolds influences metastatic progression systemically through a foreign body reaction (FBR). In this study, we integrated the concept of creating an artificial niche to capture tumour cells actively disseminating in the peritoneal cavity with a therapeutic strategy modulating the interactions of metastatic cells with the ECM. The aim was to transform a disseminated disease into a focal disease. For this, we designed and developed a ‘biomimetic’ ECM composed of a nonresorbable three-dimensional scaffold with collagen coating and characterized the FBR to the implanted biomaterial. We also analysed the safety of the implanted devices and their ability to capture tumour cells in different murine preclinical models of advanced ovarian cancer. Implantation of the biomimetic devices resulted in an initial inflammatory reaction that transformed progressively into a fibrous connective tissue response. The adhesive capabilities of the scaffold were improved with the ancillary effect of the FBR and showed clinical utility in terms of the efficacy of capture of tumour cells, disease focalization and survival benefit. These results demonstrated the performance and safety of this ‘biomimetic’ ECM in preclinical models of advanced ovarian cancer. Translated into the clinical setting, this new therapeutic strategy represents the possibility for control of peritoneal carcinomatosis upon primary ovarian debulking surgery and to expand the percentage of patients who are candidates for second rescue surgeries at the time of relapse. Editor's choice: A new therapeutic strategy targeting peritoneal metastasis based on biomimetic devices that capture disseminating tumour cells is used as a proof of concept in clinically relevant models of advanced ovarian cancer. The concept of ‘pre-metastatic niches’ refers to the conditioning of future sites of metastasis or ‘soil’ in preparation for the reception of tumour cells, and the crosstalk of tumours with the environment at distant sites to improve the efficiency of the process of metastasis . A combiAmong gynaecological malignancies, ovarian cancer is usually diagnosed at an advanced stage, when tumours have spread in diffuse peritoneal lesions that impede surgical removal. Nowadays, the survival rate at 5 years in advanced ovarian cancer does not exceed 25% . The bioA new cancer cell capture technology was recently developed to redirect peritoneal metastasis by offering an optimal microenvironment that competes with the natural niches of tumour cell homing. The initial design combined an implantable scaffold coated with exosomes as the capture agent, resulting in tumour cell attachment as its nonpharmacological mode of action . Extracen=6; n=6; P=0.002; n=6; The impact of the known FBR described in association with porous synthetic biomaterials on the efficacy of tumour cell capture of bare nonresorbable scaffolds was studied in a murine model of ovarian cancer peritoneal dissemination. The scaffolds were surgically implanted at the inner wall of the peritoneum, and the capture efficacy of 1 million intraperitoneally injected luciferase-SKOV3 ovarian cancer cells was evaluated with time. In the absence of any device, sacrificed mice presented a pattern of peritoneal metastasis with the fat surrounding the diffuse pancreas and the uterine fat as the natural sites for luciferase-SKOV3 cell implantation A. The imin vivo homing of tumour cells, the production of ECM proteins would improve the capture efficacy of the bare scaffolds. We thus designed a biomimetic device composed of a nonresorbable scaffold with a type I collagen coating . The additive contribution of each of the two elements of the biomimetic device (scaffold+coating) was confirmed both in vitro in an orbital adhesion assay, which mimics peritoneal dissemination in ovarian cancer , and in vivo, reproducing the complete remodelling of the pattern of peritoneal dissemination. The peritoneal metastases were localized in a unique location within the biomimetic device 1 week after implantation (n=8), an improved adhesive capacity compared with 63.6% for the bare scaffold . Biomimetic devices from the high-collagen group with the targeted collagen coating level designed for clinical use (250 mg) captured 100% of SKOV3 tumour cells injected . We have also demonstrated the efficacy of the biomimetic technology to capture other clinically relevant tumour cells disseminating in the peritoneal cavity, independent of their tumour origin. Tumour cells lines representative of carcinoma serous and endometrioid histology, in addition to patient-derived ovarian tumour cells, were effectively captured by the biomimetic devices with the targeted collagen coating level designed for clinical use, resulting in the transformation of a peritoneally disseminated disease into a focal disease . Together, these findings support the adhesive, nonpharmacological mode of action of the device, with the principal tumour cell capture action provided by the bare scaffold and an ancillary improved capture efficacy provided by the collagen coating, overall recapitulating a metastatic niche.Given that this FBR characterized by fibroblast-to-myofibroblast transition is associated with matrix deposition and elevated collagen synthetic capacity that results in fibrosis, we speculated that in addition to the reticulated structure of the scaffolds favouring the antation F. Quantiantation G. In theantation G; n=8, aP=0.033; G; n=8. BP=0.004; G; n=8. WFig. S4). This supports the long-term effectiveness of the biomimetic device, with 97.7% of captured tumour cells at 1 month (n=4), 94.3% at 6 months (n=12) and 93.3% at 12 months (n=6). No statistically significant differences in the efficacy of tumour cell capture by the biomimetic device were observed among the three time points .We next characterized the host tissue response generated by the biomimetic devices intended for clinical use. Haematoxylin and Eosin (H&E) staining indicated an acute inflammatory response characterized by the presence of polymorphonuclear cells at short times after device implantation , also indicating that capture of tumour cells by the device does not promote tumour proliferation and does not affect overall tumour burden but does influence the pattern of metastasis. We also demonstrated that the biomimetic device did not contribute to or promote any advantageous proliferative effect on the captured tumour cells, in an in vivo model of subcutaneous tumour growth . Consistently, we also demonstrated that once tumour cells disseminating in the peritoneal cavity had been captured by the device, no dissemination or release of these captured tumour cells occurred from the device . The ability of the biomimetic technology permanently to retain the tumour cells that have been captured by the device precludes any associated risk of promoting new peritoneal metastasis upon tumour cell capture.After characterizing the mode of action of the biomimetic technology and the impact of the FBR, we assessed the efficacy of tumour cell capture in a murine model of advanced ovarian cancer mimicking a recurrent disease setting with sustained release of tumour cells from existing peritoneal metastases. For this, we implanted the biomimetic devices after the formation of tumour implants at the natural sites of metastasis, pancreas and gonadal fat pad (post-injection model), by intraperitoneal injection of luciferase-SKOV3 cells (left panel in n=8). In the biomimetic group, the devices were surgically implanted in the inner peritoneal wall of mice, and, 1 week after surgery, tumour cells were injected intraperitoneally . This group represented survival benefits attributable to the focalization of the peritoneal disease. In a second scenario intended to mimic recurrent disease, the devices were surgically implanted 1 month after intraperitoneal injection of tumoural cells . In a third scenario, the devices were implanted, and tumour cells were injected intraperitoneally 1 week after surgical implantation. One month after injection of tumour cells, the devices were surgically removed . This group represents survival benefits attributable to surgical removal, the intended clinical use of the device.The biomimetic device has been designed for use in a clinical setting in conjunction with current therapeutic strategies, including surgery and chemotherapy, with its clinical success being based on its ability to transform a systemic disease into a localized disease. We assessed the impact on survival outcomes in a preclinical study that simulated its intended clinical use , but demonstrated the ability of the devices to remodel the pattern of peritoneal metastasis .Once the study endpoint was reached, defined as a decrease in the performance status of the mice, according to the Directive 2010/63/EU guideline, the animal was sacrificed. Bioluminesce images illustrate the focalization of peritoneal disease within the device in the biomimetic group, and within the device in addition to the pancreas and gonadal fat pad in the biomimetic post-injection group, in comparison to massive peritoneal carcinomatosis in the control group B. Finalltastasis B. Also, P<0.0001; green line in P=0.0005; blue line in P<0.0001; red line in The impact of tumour cell capture and the consequent focalization of peritoneal carcinomatosis on survival outcome is illustrated in the Kaplan–Meier survival curves C. Mice iHistological analysis of peritoneal tissues confirmed that the presence of the biomimetic devices resulted in a significant reduction of the massive peritoneal carcinomatosis shown in mice in the control group, with macro-metastasis in the pancreas and gonadal fat pad as the natural sites of metastasis in all the animals included in this group, and both macro- and micro-metastasis in other peritoneal tissues in five out of eight animals D. A reduAll these findings demonstrate a statistically significant benefit in mean survival outcomes resulting from use of the biomimetic devices in three different clinical scenarios. This is especially relevant in the biomimetic removal group , as representative of the planned use of the device in clinical practice.Metastatic tumour cell growth and survival in distant organs is facilitated by the formation of a pre-metastatic niche that is basically composed of haematopoietic cells, stromal cells and ECM. The creation and evolution of these niches is supported by existing tumour lesions through complex systemic communication involving extracellular vesicles, and results in an improved efficiency of the homing of circulating tumour cells and micro-metastasis formation . Likewisin vivo, thus providing an ectopic site for the recruitment of metastatic tumour cells. In addition to the tumour cell capture action demonstrated by the bare porous scaffold, the FBR associated with the recruitment of immune cells and fibroblasts provided additional capture abilities to the device, as shown by the progressive improvement in efficiency correlating with collagen deposition. The definitive design of the biomimetic technology includes the coating of the scaffold with collagen, resulting in a complete and durable, efficient tumour cell capture ability of the biomimetic technology from the moment of implantation in advanced preclinical models of the peritoneal dissemination of ovarian cancer.The host response to an implanted biomaterial includes the formation of a fibrous capsule consisting of inflammatory immune cells and fibroblasts around the border of the implant. As also demonstrated in this work, tumour cells can home in to an implant owing to the local foreign body response alone. The composition of the immune cells in the foreign body response might differ depending on the host, modulating the formation of a pre-metastatic niche at an ectopic location . In addiFig. S9). Alternatively, neoadjuvant chemotherapy followed by interval debulking surgery can be performed to reduce tumour volume and improve resectability, but, despite radical surgery and chemotherapy, most patients with ovarian cancer develop recurrence and die owing to progressive disease ], is treated by cytoreductive surgery to eliminate the intra-abdominal tumour burden followed by platinum- and taxane-based chemotherapy . To this disease . Relapse disease .Fig. S10). In addition, the presence of the devices and their ability to capture tumour cells preferentially could also serve to identify metastatic disease at the earliest stage for patients at risk of recurrence, enabling the initiation of therapy while the disease burden is low  before its translation into clinical use. Likewise, the impact of a complete set of immune components, limited in this work by the use of immunocompromised preclinical models, should be evaluated, although preliminary results indicate that the efficacy of tumour cell capture is maintained in immunocompetent models ; RRID:CVCL_M089] and maintained in McCoy's 5A complete medium . This human cell line has been authenticated using short tandem repeat profiling within the past 3 years. All experiments were performed with mycoplasma-free cells. million cells were injected intraperitoneally.Female severe combined immunodeficient (SCID) CB-17/lcr-Prkdcscid/scid/Rj mice  aged 6-8 weeks were used. Mouse models of advanced ovarian cancer were generated by intraperitoneal injection of 100 μl PBS containing 1 million SKOV3-luciferase cells in the peritoneal cavity; alternatively, for survival studies, 2.5Fig. S1; MTrap, Inc., Huntington, NY, USA). Collagen is the most abundant protein of the ECM, and type I collagen, in particular, is a well-established cell adhesion molecule with a long history of use in regenerative medicine applications. The polycarbonate polyurethane scaffold is a nonresorbable, reticulated, three-dimensional scaffold that has been shown in preclinical studies to serve as an effective bioengineered bone marrow niche . The wound was closed using Ethicon VICRIL®  suture. Isoflurane  at a concentration of 2% isoflurane in air was used for general anaesthesia. Buprenodale  was used for postoperative analgesia by subcutaneous injection at 1 mg/kg.Biomimetic devices were surgically implanted through a midline ventral incision. The dimensions of the preclinical devices (6 mm×3 mm×2 mm) were designed based on an eventual surgical laparoscopic implantation of scaled clinical biomimetic devices in a future clinical trial. The maximal concentration of the collagen coating was determined by assessment of device permeability and scanning electron microscopy to achieve acceptable collagen coverage throughout the porous scaffold structure without compromising permeability, supporting cellular infiltration and proliferation (data not shown). The device was fixed to the mesothelium opposite to the pancreas on the inner wall of the peritoneum using a surgical glue  coupled to Living Imaging software v.4.2 (Xenogen) was used to detect the pattern of peritoneal metastasis by bioluminescence imaging. Luciferin  was used as the substrate for the luciferase-expressing tumour cells and injected intraperitoneally at a concentration of 150 mg/kg in PBS. The bioluminescence signal was used to quantify the number of tumour cells as normalized photons within each study, and a corresponding colour scale was used to represent them in the Tissue samples and removed devices were immersion fixed in 10% buffered formalin for 24 h, dehydrated in an ethanol series, embedded in paraffin, sectioned (3 μm thick) and mounted on microslides. After drying, dewaxing and rehydratation, tissues were stained with H&E and examined using a LEICA DMD108 digital microscope . The histological analysis of the FBR at the different time points was performed by two independent pathologists, and a minimum of three random fields were evaluated using a semi-quantitative score that considered the amount of inflammatory cells , the presence of erythrocytes, haemosiderin deposition, mineralized deposits and the amount of collagen deposition and scored these as follows: (−)=nil, (+)=mild, (++)=moderate and (+++)=marked. Alternatively, the histological analysis of micro- and macro-metastases in the survival study was performed in at least three H&E sections processed from the paraffin block at intervals of 30 µm and examined by two independent pathologists. Tissues positive for the presence of metastasis were evaluated further to determine the size of each metastatic nodule measured with a glass millimetre ruler and classified as micro-metastases (measuring ≤5.0 mm) or macro-metastases (measuring >5.0 mm).Formalin-fixed, paraffin-embedded tissue blocks sectioned at 3 μm thickness were dried for 15 min at 65°C before the staining procedure with the Artisan™ Link Special Staining System .Formalin-fixed, paraffin-embedded tissue blocks were sectioned at a thickness of 3 μm and dried for 1 h at 65°C before the pretreatment procedure of deparaffinization, rehydration and epitope retrieval in the Pre-Treatment Module, PT-LINK (Agilent Technologies-DAKO) at 95°C for 20 min in 50× Tris/EDTA buffer, pH 9, for anti-LYVE1 antibody, and at 95°C for 20 min in 50× citrate buffer, pH 6 for anti-collagen type I antibody . Before staining the sections, endogenous peroxidase was blocked. After incubation with the primary antibody, the reaction was visualized with the EnVision™ FLEX Detection Kit (Agilent Technologies-DAKO) for collagen I and with Biotin-SP-AffiniPure Goat Anti-Rabbit IgG  and streptavidin  for collagen type I. Diaminobenzidine chromogen was used as a substrate. Sections were counterstained with Haematoxylin.t-test was used to compare the differences between two groups, and analysis of variance was used to compare the differences among multiple groups. Kaplan–Meier survival curves were used to estimate cumulative survival probabilities using IBM SPSS statistics. The overall P-value was calculated using a log-rank test. Statistical significance was set at P<0.05.The normalized bioluminescence/fluorescence signal at the area of the biomimetic device, compared with that of the rest of the peritoneal tissues, was used to evaluate the efficacy. All experiments were repeated at least three times. Student's


Results show that the probes display evident fluorescence enhancement when bound to the β-amyloid, suggesting the effect of the environment on the molecular properties. Especially, the two-photon absorption cross-section of the probes increase drastically in the β-amyloid compared to that in vacuum, which results from the larger electron delocalization and dipole moment in the fibrillary-like environment. Thus, one can propose that the studied probes are capable of application in two-photon fluorescent imaging, particularly those containing naphthalene rings as the donor or with a longer spacer group. Our calculations elucidate the experimental measurements reasonably, and further establish possible structure–property relationships that can be used to design novel biocompatible two-photon fluorescent probes for the diagnosis of Alzheimer’s.Alzheimer’s disease (AD) is one of the most common forms of senile disease. In recent years, the incidence of AD has been increasing significantly with the acceleration of the aging process of the global population. However, current clinical drugs can only alleviate the symptoms of AD patients without healing the disease fundamentally. Therefore, it is of great significance to develop an effective small molecule diagnostic reagent for the early diagnosis of AD. In this paper, we employ an integrated approach, including molecular docking simulation and quantum mechanics/molecular mechanics calculation, to investigate the sensing performance of a series of donor–acceptor structural probes for the marker protein of AD ( In early stages of the disease, memory loss of the patient is mild, but at late stages, individuals will lose the ability to carry on a conversation or respond to the environment ,2. Accorms occur ,6. Thus,heimer’s ,8.In comparison to the probes based on radionuclide imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), a near-infrared fluorescent (NIRF) probe shows advantages on the safety, accuracy, sensitivity, as well as operability ,10,11. Nβ-amyloid imaging is to explore the two-photon properties of the dyes that have already shown some success in this aspect. Up to now, plenty of fluorescent probes with multiple chemical structures have been synthesized and experimentally applied to detect the β-amyloid plaques in vivo, such as PAD-1, DCIP-1, and BAP-1 with donor–acceptor architecture [N,N-dimethylamino group as the donor and dicyano group as the acceptor to study their possibility to be two-photon fluorescent probes for detecting β-amyloid.A potential route to identify efficient two-photon sensors for itecture ,17,18, aitecture ,20,21. Ditecture ,23, we cβ-amyloid imaging. Ono et al. selected benzothiazole as the highly polarized bridge and connected N,N-dimethylamino and dicyanomethylene to synthesize a series of push–pull dyes [β-amyloid aggregates. Before long, they evaluated the feasibility of a series of smart NIRF probes with donor–acceptor structure bridged by a conjugated π-electron chain for detecting β-amyloid [β-amyloid plaques by replacing the benzene ring with a naphthalene ring, which could readily penetrate the blood–brain barrier with a large initial brain uptake [In the past decades, several dicyano-containing dyes have been designed as NIRF probes for ull dyes . In bind-amyloid . The pron uptake .β-amyloid [β-amyloid in Alzheimer’s disease. Additionally, we aim to set out possible structure–property relationships for the future design of improved diagnostic probes by analyzing the effect of donor moieties and the length of spacers on the sensing performance of the probes. To this end, another three derivatives of the same series named P4, P5, and P6  calculations.Molecular structures of the probes studied in this paper  are shown in -amyloid ,27, our d P6 see  are inveβ-amyloid. The geometry for the probes was optimized by the density functional theory (DFT) method at the B3LYP/6-31+G level using Gaussian 16 [β-amyloid and no well-defined binding site reported previously, we employ blind docking studies with a grid box of 126 × 126 × 76 points and a spacing of 0.464 Å in AUTODOCK 4.2 software [Molecular docking was carried out to identify the potential binding sites of P1, P2, and P3 with ssian 16 . A full-ssian 16 . As thersoftware . The semβ-amyloid was treated as the MM part and defined to be the low layer  and emission (OPE) spectra of the probes in different binding sites of Last but not least, two-photon absorption (TPA) properties for the probes were calculated by employing the response theory method with the DFT/B3LYP functional and 6-31G+ basis set using Dalton2013 software . In ordeβ-amyloid, as demonstrated in β-amyloid are shown in β-amyloid. Furthermore, in comparison to P2, probe P3 with a longer polyenic chain possesses a lower binding free energy. Thus, the length of the π-conjugated bridge between the donor and acceptor units also contributes to molecular binding affinity.Molecular docking simulations on the probes show that there are two most likely binding sites for each probe toward Apart from the binding energy, the inhibition constant is another important parameter to evaluate the bioactivity of an agent. The calculated data from molecular docking are listed in β-amyloid, one can expect that molecular photophysical properties may feature a combined effect over contributions from different sites. We computed OPA spectra for the probes in different binding sites, and the spectrum in vacuum is also shown as a reference in β-amyloid, which are located at wavelengths around 350 and 550 nm, respectively. In view of multiple binding modes found for the probes toward An analysis of molecular orbitals involved in the OPA process shows that the absorption band in the long wavelength range is associated with the transition from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) of a probe. Frontier molecular orbitals for the probes in different microenvironments are shown in β-amyloid, the absorption wavelength of P1 is blueshifted by 11 and 19 nm in binding site1 and site2, respectively, compared to the case in gas, indicating the microenvironment effect on the molecular optical properties. On the contrary, the maximum OPA peaks of P2 and P3 are redshifted in β-amyloid, especially for P3 in site2, and the shift is more than 30 nm. Thus, adding a double bond to the dye skeleton may result in a redshift of the maximal OPA wavelength of these probes. As it has been reported, the experimental absorption band maxima are 557 nm for P2 and 559 nm for P3 [Details of the OPA peak with a wavelength larger than 500 nm for the probes, including absorption wavelength, the corresponding oscillator strength, and transition nature, are listed in m for P3 . Even thβ-amyloid, except for the case of P3 binding in site2. According to the equation fω and αμ denote the excited energy of the state |f > and electric dipole moment operator in the direction α, respectively, OPA strength is dominated by the transition dipole moment between the ground state and the excited state. Calculated transition dipole moments for P1, P2, and P3 are 3.17, 3.56, and 3.91 Debye in vacuum, respectively. Within β-amyloid, molecular transition dipole moments are increased for P1 and P2 both in site1 and in site2. However, for P3 in site2, the value is reduced to 3.63 Debye compared to that in gas, resulting in a smaller oscillator strength.In addition to the shifts in OPA spectra, the absorption intensity for the probes is increased to different degrees when bound to |f〉|2)/3 , where ωβ-amyloid as a fluorescent sensor, OPE properties of the probes in different microenvironments are investigated by optimizing the molecular geometry in the first excited state using the QM/MM method. One can see from the emission spectra in β-amyloid. This off–on transformation on the fluorescent signal of the probes appears to be in good agreement with the experimental observations. Within β-amyloid, the maximum emission wavelengths for P1 in site1 and site2 are respectively 600 and 591 nm. By replacing the donor moiety with benzothiazole in P1, the emission maxima are shifted to 593 nm (589 nm) for P2 and 614 nm (656 nm) for P3 in site1 (site2). Although these values are smaller than the experimental results of 660 nm for P2 and 710 nm for P3 [β-amyloid and nuclear motion of the probe that are not included in our treatment.To investigate the feasibility for the dyes to detect m for P3 , the lenm for P3 . In geneTransition properties of the first excited state play an important role in determining the fluorescent characteristics. Thus, analysis of the nature of excitation corresponding to the emission peak is performed. It shows that the fluorescence of the probes in amyloid comes from the molecular first excited state radiation, which is predominated by the LUMO-to-HOMO transition. As shown in β-amyloid are displayed in β-amyloid, changes in the geometries between the ground state and the first excited state of the probes are not obvious, because the rotation of the groups is hindered by the amyloid. To quantitatively characterize the geometric variations between the ground state and the first excited state of the probes, the root-mean-square displacement (RMSD) for the two states in different microenvironments is calculated using Multiwfn [β-amyloid, the values are smaller than 0.1 Å, showing a slight change in the molecular configuration in different states. In conclusion, the probes show off–on fluorescence when they are bound to the amyloid, indicating the availability of P1, P2, and P3 as fluorescent probes for detecting β-amyloid in Alzheimer’s disease.In order to rationalize the changes in molecular orbitals’ distribution, geometries of the probes both in gas and in Multiwfn . In the β-amyloid imaging. The macroscopic TPA cross-section (TPAσ) that can be directly compared to the experiment is defined as [Motivated by the results above, we next investigate the probability of the probes as a two-photon fluorescent sensor for fined as (1)σTPA=a0 is the Bohr radius, c is the speed of light, α is the fine structure constant, ω is the frequency of the incident light, and g(ω) denotes the spectral line profile, which is usually assumed to be a delta function. The level broadening Γf of the final state is commonly assumed to be 0.1 eV, corresponding to a lifetime of about 6 fs. TPAδ in the above equation denotes the microscopic TPA cross-section. When excited by linearly polarized light, the expression of TPAδ can be written as [Here, itten as (2)δTPA=essed by (3)Sαβ=∑i> including the ground state |0> and the final state |f>, ιω is the corresponding intermediate state energy, and α(β)μ is the dipole moment operator in the direction α,β ∈ .Here, the summation goes over all intermediate states |β-amyloid are presented in β-amyloid (411 to 166 GM in the gas phase) going from P1 to P2, showing the influence of donor moieties on the two-photon response of the probes. However, the value of the maximum TPA cross-section possesses a nearly threefold enhancement in P3 compared to P2, corresponding to an increase in the TPA performance upon spacer elongation. Particularly, the maximal TPA wavelength for the probes is located in the near-infrared region, which indicates the possibility of probing a deeper region in practice. Moreover, the TPA wavelength maximum of the probes is comparable to the value of 1000 nm for an experimentally reported two-photon β-amyloid sensor [Calculated TPA cross-sections for the probes both in the gas phase and in different binding sites within d sensor , which cx direction and short axis of the molecule as the y direction. As can be seen in xxS in different microenvironments, which corresponds to the transition along the molecular macroaxis. When the probes are bound to β-amyloid, the value of xxS increases sharply, indicating an enhancement in transition dipole moment between the ground state and the TPA state of the probe according to Equation (3). Moreover, the magnitude of xxS for different probes follows the order P1 > P3 > P2 both in vacuum and in β-amyloid, showing the same trend as the maximum TPA cross-section. One can thus achieve the conclusion that the substitution of the benzothiazole moiety or lengthening of the π bridge in P2 would effectively improve the electron delocalization and transition dipole moment of the molecule, further enhancing the TPA transition probability. The above theoretical investigations reveal the inner perspectives for the usage assumption on the probes to be a practicable two-photon imaging chemosensor for detecting β-amyloid plaques in Alzheimer’s disease.As is known, the TPA cross-section is dominated by the two-photon transition matrix element. Here, we set the long axis of a molecule as the β-amyloid are analyzed by using an integrated approach including molecular docking and QM/MM calculations. Results demonstrate that these probes show evident fluorescence enhancement when bound to the amyloid, indicating the key role of the environmental effect on molecular properties. Furthermore, an increased molecular TPA cross-section is found in the β-amyloid compared to that in the gas phase, which results from the large electron delocalization and dipole moment. We arrive at the conclusion that the studied probes can be applied for the two-photon imaging technique, particularly the ones containing a naphthalene ring as the donor and with a longer spacer group. Our calculations give a reasonable explanation for the previous experimental observations, and provide information on the molecular structure–property relationship, which is beneficial for developing new efficient fluorescent probes toward β-amyloid.Encouraged by the good qualities of two-photon excited fluorescent imaging, we have studied a series of donor–acceptor dyes for their usage in Alzheimer’s early diagnosis as a two-photon fluorescent probe. The binding profiles and photophysical properties of the probes in