N,N-dimethylacrylamide, diallyldimethyl ammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid, maleic anhydride and chemical crosslinking agent triallylamine through free radical copolymerization. Due to the synergistic effect of covalent micro-crosslinking and the reverse polyelectrolyte effect of amphoteric polymers, the copolymer-based drilling fluids exhibit outstanding rheological and filtration properties even after aging at high temperatures (up to 200 °C) and high salinity  environments. In addition, the zeta potential and particle size distribution of copolymer-based drilling fluids further confirmed that the copolymer can greatly improve the stability of the base fluid suspension, which is important for reducing the fluid-loss volume of WDFs. Therefore, this work will point out a new direction for the development of temperature- and salt-resistant drilling fluid treatment agents.With increasing global energy consumption, oil/gas drilling has gradually expanded from conventional shallow reservoirs to deep and ultra-deep reservoirs. However, the harsh geological features including high temperature and high salinity in ultra-deep reservoirs have become a critical challenge faced by water-based drilling fluids (WDFs), which seriously deteriorate the rheology and fluid loss properties, causing drilling accidents, such as wellbore instability and formation collapse. In this study, a novel temperature- and salt-resistant micro-crosslinked polyampholyte gel was synthesized using  Drilling fluid or mud, known as the “blood” of drilling engineering, plays vital roles in entire well-drilling operations, such as cleaning the borehole, suspending and carrying drill cuttings, balancing the pressure between the formation and the wellbore, cooling and lubricating the drill bit, etc. ,2,3,4,5.As the main additive of WDFs, fluid loss control additives with excellent temperature- and salt-resistance performance are very important in controlling the fluid loss of drilling fluid and stabilizing the wellbore, having attracted attention from academia and industry ,17,18. TAs a comparison, synthesized polymer, with the advantages of adjustable functional groups, adjustable group ratio, regular molecular chain structure, and controllable molecular chain length, has been the mainstream development direction of temperature- and salt-resistant fluid loss agents in the past 20 years ,27. GeneN,N-dimethylacrylamide (DMAA), cationic monomer diallyldimethyl ammonium chloride (DMDAAC), anionic monomer 2-acrylamido-2-methylpropanesulfonic acid (AMPS), maleic anhydride (MA) and chemical crosslinking agent triallylamine through free radical copolymerization, as shown in Herein, a novel high temperature- and salt-resistant micro-crosslinked polyampholyte gel  was synthesized using 1H NMR spectroscopy, FTIR and elemental analysis of DDAM were performed. As shown in 2–CH–CO– (in DMAA or AMPS), –CH–CO– (in MA) and –CH2–CH– (in DMDAAC) groups were at the regions of 1.25–1.75 ppm. The CH3– proton peaks in DDAM appeared at 3.0 ppm. The C–2(CH3)– and –CH2–SO3– in AMPS could be observed at 1.0 ppm and 3.1 ppm, respectively. Peak (3.0–3.3 ppm) should be attributed to the CH3–N+ and –CH2–N+ in DMDAAC. As a result, the above characteristic peaks of atomic groups demonstrated the successful synthesis of the copolymer. In addition, the FTIR spectra of DDAM was shown in −1 was caused by the asymmetric stretching vibration of CH2 in the main chain of the copolymer. The peaks at wavelengths of 1668 and 1456 cm−1 were caused by the carbonyl C=O in the amide group and the stretching vibrational peak of –CN– in DMAA, respectively. The broad peak at a wavelength of 2242 cm−1 was attributed to the stretching vibration peaks of the carbonyl C=O in MA. The peaks at 1552 and 1400 cm−1 were attributed to the -NH bond on the amino group and the vibrational absorption peak of the five-membered heterocyclic –CN– in DMDAAC, respectively. The peak at 626 cm−1 corresponded to the absorption peak of –CS in AMPS while the peaks at 1214 and 1058 cm−1 were attributed to the stretching vibrations of the sulfonic acid groups in AMPS. Besides, the characteristic peaks of unsaturated olefin bonds in DMAA, MA, AMPS and DMDAAC monomers did not appear on the FTIR spectral, indicating that the monomer reaction was complete. Moreover, as shown in In order to clarify the structure of DDAM, 2 atmosphere, and the mass loss of the polymer could be divided into four stages  and differential thermogravimetric (DTG) curves of DDAM were performed in a Nr stages d. The fiSince the rheology of the drilling fluid is essential for carrying and suspending cuttings, as well as maintaining the stabilization of the wellbore, the rheological properties of the DDAM solutions and DDAM-based drilling fluid were analyzed, respectively. As shown in Further, as shown in + destroyed the electrostatic force between the carboxyl group, sulfonic acid group and quaternary ammonium group, and reduced the degree of folding and curling of the polymer chain, which lead to the increase of polymer hydrodynamic volume, thus increasing the viscosity of the drilling fluid.Usually, salt would affect the performance of WDFs from the following two aspects: one is the metal cations adsorbed on the surface of the bentonite would compress the electric double layer and decrease the electrostatic repulsion between bentonite, which causes the agglomeration of bentonite; on the other hand, the salt would cause the polymer chain to curl, which weakens the interaction between the polymer and the bentonite, thus increasing the particle size and the fluid loss of the drilling fluid. In order to test the salt pollution resistance of drilling fluid, the influence of electrolytes on the rheological properties of drilling fluid based on 2 wt% DDAM content was studied. As shown in Driven by the positive pressure difference between the mud static column pressure and the formation wellbore pressure, the liquid part, as well as solid particles of the drilling fluid, would permeate into the formation through the wellbore cracks, gradually forming a low permeability mud cake on the wellbore, which was called the filtrate loss and wall-building functions of drilling fluid. The fluid loss performance of drilling fluid mainly referred to the fluid loss volume of the drilling fluid and the quality of the formed mud cake, which was very important to maintain the stability of the wellbore. For the evaluation of fluid loss of drilling fluid, the American Petroleum Institute (API) standard states were usually used. It could be seen from + would compress the electric double layer between the bentonite particles, which weakens the hydration and dispersion of the bentonite, resulting in poor quality of the mud cake and increased fluid loss. When 5 wt% NaCl was added to the drilling fluid containing 2 wt% DDAM, the thickness of the mud cake increased to 1.5 mm  even after aging at high temperatures (up to 200 °C) and high salinity  environments. Moreover, the zeta potential and particle size distribution of DDAM-based drilling fluids further confirmed the enhanced rheological and filtration properties could be attributed to the presence of positive and negative functional groups which could effectively be adsorbed on the bentonite particles and inhibit the aggregation of the bentonite particles. Overall, this work can promote the application of micro-crosslinked polyampholyte gel as a fluid-loss additive for WDFs under extremely high temperature and high salinity conditions.In this work, a novel high temperature- and salt-resistant micro-crosslinked polyampholyte gel filtration reducer  was synthesized using DMAA, DMDAAC, AMPS, MA and chemical crosslinking agent triallylamine through aqueous free-radical copolymerization. TGA analysis demonstrated the DDAM copolymer exhibited excellent thermal stability and had great potential for application in high temperature resistant drilling fluids. Meanwhile, due to the reverse polyelectrolyte effect, DDAM still shows the ability to increase viscosity in high saltwater environments. As a result, when 2 wt% DDAM was added to the base drilling fluid, the DDAM-based drilling fluids exhibited outstanding rheological and filtration properties , diallyldimethyl ammonium chloride , 2-acrylamido-2-methylpropanesulfonic acid , maleic anhydride  were purchased from Sigma-Aldrich Corporation . Initiator ammonium persulfate , chemical crosslinking agent triallylamine (99 wt%), and sodium hydroxide  were obtained from J&K Chemical, Ltd. . Bentonite (sodium form) was from Weifang, Shandong. All reagents were used as received without further purification unless specified.Monomers First, 12.38 g DMAA, 7.35 g MA, 10.35 g AMPS, 8.05 g DMDAAC and 0.20 g chemical cross-linking agent triallylamine  were dissolved in deionized water (pH adjusted to 8~9 with sodium hydroxide), and the total mass fraction of reactive monomers was adjusted to 30 wt%. Secondly, the above solution was put into a three-necked flask, stirred at high speed for 30 min under the protection of nitrogen, and heated to the reaction temperature (60 °C). Finally, 0.08 g ammonium persulfate initiator  was added to initiate the polymerization reaction for 8 h. After the reaction, the solution was washed with a mixed solution of absolute ethanol and acetone, precipitated, and filtered to obtain a micro-crosslinked polyampholyte gel fluid loss control agent (DDAM).2O) as the solvent. Fourier transform infrared (FTIR) spectrometry (A225/Q Platinum ATR) was also conducted to confirm the structure of DDAM. The elemental composition of DDAM was analyzed by the Elementar Vario EL instrument. The thermal property of DDAM was evaluated by the thermogravimetric analyzer  under nitrogen purging and a heating rate of 10 °C/min.NMR spectra of DDAM were acquired using an NMR spectrometer (Bruker AVANCE III NMR) with deuterated water  was dissolved in 400 mL of base drilling fluid with 8000 rpm stirring for 20 min. For salt contamination, different mass fractions of sodium chloride were slowly added into the DDAM-based drilling fluid at 5000 rpm stirring for 20 min. In order to test the temperature- and salt-resistance of the WDFs, the above-mentioned salt-contaminated DDAM-based drilling fluid was added to a roller oven (Fann Instrument Company) and aged at different temperatures for further trials.The API filtrate performance test was measured by using a ZNZ-D3 medium pressure filtration loss apparatus . The volume of a certain volume of fluid filtered through filter paper (9 mm diameter and 2.7 μm pore size) for 30 min under a pressure of 0.7 MPa was the API filtrate volume.−1 and the frequency was 1 Hz. In addition, the rheology of DDAM-based drilling fluid was measured according to the American Petroleum Institute (API) standards. Firstly, test the dial reading Ф600 (600 r/min) and Ф300 (300 r/min) by a six-speed rotating viscometer . Thereafter, calculate the apparent viscosity (AV), plastic viscosity (PV), and yield point (YP) from the viscometer test data:The rheology of the DDAM solution was measured by a rotary rheometer . The elastic and viscous moduli of DDAM solution as a function of strain were obtained by the oscillatory shear mode . The viscosity of the DDAM solution was measured under the shear rate from 1 to 500 s600 (mPa·s)Apparent Viscosity (AV) = 0.5Ф600 − Ф300 (mPa·s)Plastic Viscosity (PV) = Ф300 − 0.5Ф600 (Pa)Yield Point (YP) = Ф


Among critically ill patients with acute kidney injury (AKI), earlier initiation of renal replacement therapy (RRT) may mitigate fluid accumulation and confer better outcomes among individuals with greater fluid overload at randomization.We conducted a pre-planned post hoc analysis of the STandard versus Accelerated initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial. We evaluated the effect of accelerated RRT initiation on cumulative fluid balance over the course of 14 days following randomization using mixed models after censoring for death and ICU discharge. We assessed the modifying effect of baseline fluid balance on the impact of RRT initiation strategy on key clinical outcomes. Patients were categorized in quartiles of baseline fluid balance, and the effect of accelerated versus standard RRT initiation on clinical outcomes was assessed in each quartile using risk ratios (95% CI) for categorical variables and mean differences (95% CI) for continuous variables.p = 0.03). Accelerated RRT initiation did not confer greater 90-day survival in any of the baseline fluid balance quartiles (quartile 1: RR 1.11 (95% CI 0.92 to 1.34), quartile 2: RR 1.03 (0.87 to 1.21); quartile 3: RR 1.08 (95% CI 0.91 to 1.27) and quartile 4: RR 0.87 (95% CI 0.73 to 1.03), p value for trend 0.08).Among 2927 patients in the modified intention-to-treat analysis, 2738 had available data on baseline fluid balance and 2716 (92.8%) had at least one day of fluid balance data following randomization. Over the subsequent 14 days, participants allocated to the accelerated strategy had a lower cumulative fluid balance compared to those in the standard strategy  versus 5646  mL, Earlier RRT initiation in critically ill patients with AKI conferred a modest attenuation of cumulative fluid balance. Nonetheless, among patients with greater fluid accumulation at randomization, accelerated RRT initiation did not have an impact on all-cause mortality.Trial registration: ClinicalTrials.gov number, https://clinicaltrials.gov/ct2/show/NCT02568722, registered October 6, 2015.The online version contains supplementary material available at 10.1186/s13054-022-04229-0. The administration of fluid is a crucial part of resuscitation in critical illness but at some point, fluid administration may become deleterious to patients as pathologic fluid overload impairs the function of multiple organs . Fluid aThe STandard versus Accelerated initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial was an international multicenter trial that compared two strategies of renal replacement therapy (RRT) initiation in patients with severe AKI who had no conventional indications for urgent RRT initiation. Patients were allocated to an accelerated strategy, which entailed RRT initiation shortly after meeting trial eligibility or to a standard strategy of structured RRT deferral until an AKI-related emergency occurred or another requirement for RRT arose based on the judgement of the attending clinician . In thisThe STARRT-AKI trial enrolled 3019 critically ill adults with severe AKI  at 168 centers in 15 countries . Key excThis planned secondary analysis aimed to evaluate the effect of an accelerated strategy of RRT initiation on fluid accumulation. First, among the 2927 patients who were eligible for the modified intention to treat analysis, we evaluated the difference in cumulative fluid balance, by randomized group, from the day of randomization through their entire ICU stay or up to day 14. We analyzed the intergroup differences in cumulative fluid balance within the following subgroups: male versus female, Simplified Acute Physiology Score (SAPS) II score > 58 versus ≤ 58, septic versus non-septic, estimated baseline glomerular filtration rate (eGFR) < 45 versus ≥ 45 mL/min/1.73 m2, medical versus surgical admission categories and geographic regions . As a sensitivity analysis, we repeated this assessment by considering only patients who received the RRT strategy to which they were allocated in addition to performing an as-treated analysis whereby patients who crossed over from their allocated arm to the opposite arm were analyzed based on the RRT initiation strategy that they actually received. In all cases, patients were censored at the time of death or ICU discharge.We divided the trial’s intention to treat cohort into quartiles based on participants’ fluid balance at the time of randomization, which reflected the balance of all inputs and outputs from the time of ICU admission until randomization. We then evaluated whether baseline fluid balance modified the effect of RRT initiation strategy on all-cause 90-day mortality  as well as selected secondary outcomes including RRT dependence in surviving patients, a composite outcome of death or RRT dependence at 90 days, and mortality in the ICU, in-hospital and at 28 days. Other outcomes of interest included ICU length of stay, hospital length of stay, ICU-free days, ventilator-free days, vasoactive-free days and hospitalization-free days. We further analyzed the modifying effect of baseline fluid balance on all-cause mortality in patients with and without sepsis and ICU admission categories . We conducted sensitivity analyses in which fluid balance at randomization was evaluated in three different ways: as deciles; as percentage fluid overload /patient’s weight) × 100) dichotomized as > or ≤ 10%; and as a continuous variable.t tests.Cumulative fluid balance was calculated on a daily basis from the day of randomization (day 0) through Day 14. Patients who left the ICU prior to Day 14 or who died were censored at the time of death or discharge from the ICU. The between-group difference in fluid balance was evaluated using a linear mixed-effects model to account for repeated measures of fluid balance for each patient. Between-group differences in cumulative fluid balance on each patient’s last day in ICU were evaluated using All trial participants who were eligible for the modified intention to treat analysis were divided into quartiles based on their fluid balance at the time of randomization. The demographic and clinical characteristics of the patients in each quartile were summarized, using means (standard deviations) or medians (interquartile ranges) for continuous variables and numbers (%) for categorical variables. To test for inter-quartile trend, linear regression models were used for continuous variables and general linear models (GLM) with binomial family and log link function for categorical variables. A logistic regression model was used to assess the interaction between fluid balance on a continuous scale and RRT strategy and the association with 90-day all-cause mortality.p value for trends was estimated as the interaction between quartile and outcome in the models. Mean differences (95% CI) for continuous outcomes were evaluated with t tests, and p values for trend were estimated with a linear regression model. For subgroup analyses, p values for the interaction between the subgroup, randomization allocation and fluid balance quartile corresponded to a 3-way ANOVA model. P values < 0.05 were considered significant. All analyses were conducted using R version 3.6.2.We evaluated effect estimates for each of the outcomes of interest within each quartile: risk ratios (95% CI) for categorical outcomes were estimated using binomial general linear models with log-links, and p = 0.03) .We evaluated 2738 patients who had fluid balance data available at the time of randomization. Of these, 2716 had at least one subsequent day of available fluid balance data. At the time of randomization, fluid accumulation was similar in patients allocated to both treatment strategies (accelerated 2581 (IQR 820–5362) versus standard 2819 (IQR 836–5603) mL). Over the subsequent 14 days, patients allocated to the accelerated strategy had a lower median cumulative fluid balance compared to patients allocated to the standard strategy  versus 5646  mL, Across subgroups, fluid accumulation was generally attenuated among patients allocated to the accelerated strategy . Patients in the highest baseline fluid balance quartile who were randomized to the accelerated arm had 4.6 (95% CI − 0.01 to 9.17) more hospital-free days. Fluid balance at the time of randomization did not modify the effect of accelerated RRT initiation on the other secondary outcomes ; quartile 2: RR 1.03 (95% CI 0.87 to 1.21); quartile 3: RR 1.08 (95% CI 0.91 to 1.27) and quartile 4: RR 0.87 (95% CI 0.73 to 1.03), p-trend 0.08) compared to the standard RRT strategy  at the time of randomization. This translates into a ~ 5% cumulative fluid balance indexed to baseline weight and is substantially lower than that observed in other cohorts of patients initiating acute RRT . Greater000 mL atThis study has several strengths. Although there have been several trials comparing RRT initiation strategies in the setting of AKI in recent years , 16–18, This work has several limitations. Though we recorded data on fluid balance at trial randomization and follow-up, we did not collect information on factors that may have affected fluid balance, notably the nature of administered fluids and use of diuretics. While our findings suggest that an accelerated strategy of RRT initiation did not impact key clinical outcomes among individuals with the greatest degree of fluid accumulation, the pragmatic nature of the trial, whereby fluid management decisions were at the discretion of treating clinicians, could have moderated any effect of the accelerated RRT initiation strategy. Finally, fluid balance was characterized by the balance of inputs and outputs from the time the patient arrived in the ICU until randomization. Though these data are widely collected in critically ill patients, they may not necessarily be an accurate reflection of pathologic organ congestion. Importantly, fluid balance measures do not include insensible losses and often do not account for fluid intake or losses prior to ICU admission. In some cases a markedly positive fluid balance is the end-result of appropriate resuscitation. For example, a patient who presents to the ICU with profound hypovolemic shock and is aggressively resuscitated in order to restore hemodynamic stability will have an appropriately high fluid balance.In a recently completed international randomized trial, an accelerated strategy of RRT initiation conferred a modest reduction in cumulative fluid balance during the two weeks following enrollment. Fluid balance at the time of randomization was associated with several markers of illness severity. However, earlier initiation of RRT did not confer improved mortality but did lead to a greater number of hospital-free days in patients with the highest degree of fluid overload. Clinical trials that evaluate optimal ultrafiltration strategies in patients receiving RRT for AKI are urgently needed.Additional file 1. Table S1: Median fluid accumulation through Day 14, in the modified intention to treat population, stratified by subgroups. Table S2: The effect of accelerated vs standard RRT strategy on outcomes across deciles of baseline fluid balance. Table S3: The effect of accelerated vs standard RRT strategy on outcomes in patients with ≤ or > 10% fluid overload at baseline.


Although serum tumour markers offer an uncomplicated, non-invasive examination method and possible therapeutic options, they are still rarely used in veterinary medicine. Our marker of interest, the Ki-67 protein, can only be detected in the active phases of the cell cycle. Therefore, it is a suitable marker for assessing the proliferating cell fraction of an organism and can thus provide information about potentially present, rapid-growing tumour tissue. The purpose of our study was to determine whether Ki-67 could be considered as a possible tumour marker in canine serum for veterinary medicine. We measured serum concentrations of Ki-67 in dogs with various malignant tumours, such as carcinomas, sarcomas, and lymphomas. In the dogs with malignant tumours we determined significantly higher serum Ki-67 concentrations compared with healthy dogs and dogs with non-malignant diseases. No significant difference in serum Ki-67 concentration was observed between the different types of cancer or between benign and malignant mammary tumours. Our investigations also included some inflammatory parameters measured in blood, such as neutrophils, lymphocytes, and monocytes, with mixed results. The results of our study suggest that Ki-67 may be useful as a potential serum tumour marker, providing information about the presence of malignant diseases in a dog.0-phase but is detectable throughout all active phases of the cell cycle. Consequently, it is used as a marker for the proliferating cell fraction of a cell population and thus could indicate neoplastic tissue present. Our study is designed to show whether Ki-67 can be considered as a potential canine serum tumour marker for veterinary medicine. We measured serum concentrations of Ki-67 in dogs with various malignant tumours (carcinomas (n = 35); sarcomas (n = 26); lymphomas (n = 21)) using a commercially available quantitative sandwich ELISA from mybiosource. Dogs with malignant tumours showed significantly higher serum Ki-67 concentrations compared to healthy dogs (n = 19) and non-neoplastic diseased dogs (n = 26). No significant difference in serum Ki-67 concentration was detected between carcinoma, sarcoma, and lymphoma, nor between mammary adenocarcinoma and adenoma. In our investigations we also included some inflammatory parameters measured in blood, such as neutrophils, lymphocytes, and monocytes, and gained mixed results. The results of our study suggest that Ki-67 may be useful as a potential serum tumour marker, providing information about the presence of malignancies in a dog.Tumour markers are scarcely used in veterinary medicine, although they are non-invasive, contribute to a faster diagnosis and new therapeutic options. The nuclear protein Ki-67 is absent in G The identification of tumour markers is a valuable investigative method in oncology. It differs from other investigative methods, such as imaging and histology, as it is less invasive, non-complex, and inexpensive to perform ,2. In huTherefore, molecules that are increasingly expressed in the context of cell proliferation are very appealing for their use as tumour markers.A promising biomarker strictly associated with cell proliferation is the nuclear Ki-67 protein, found in 1983 in a Hodgkin lymphoma cell line . The funAs a proliferation marker, Ki-67 protein is absent in resting cells, but can be identified during all active phases of the cell cycle. Throughout the interphase it can solely be detected within the nucleus, although in mitosis the majority of the Ki-67 antigen is relocated onto the surface of the chromosomes ,11.During interphase and mitosis, Ki-67 is also found to some extent in the domain of rRNA transcription, where it is considered to play an important role, one reason being that its removal inhibits ribosomal RNA synthesis ,13,14.Its selective expression in active phases during the cell cycle makes the Ki-67 protein an interesting target in cancer research. Ki-67 is already widely used in immunohistochemistry to determine the growth fraction of the cell population in question ,16. The Another contributor to tumour growth and progression are inflammatory cells, for example neutrophils, lymphocytes, and monocytes play an important role in the cancer microenvironment of tumour tissue or behave differently in it compared to a healthy organism ,18,19,20Consequently, the relationship between tumour markers, such as Ki-67, and inflammatory cells are increasingly becoming the focus of some research approaches to discover possible correlations and improve tumour diagnostics ,22.Ki-67 in itself has been investigated in humans in a variety of malignancies, especially prostate and breast carcinomas, brain tumours, nephroblastoma and neuroendocrine neoplasms ,24,25,26There are significantly less studies investigating Ki-67 as a serum tumour marker, it has been shown however, that women with malignant breast cancer have higher serum Ki-67 levels compared to control groups with non-malignant diseases ,28.To the authors’ knowledge there are no studies investigating Ki-67 in serum as cancer biomarker in veterinary medicine. In a pilot study, our group evaluated serum Ki-67 levels in a small cohort of 20 dogs with malignant tumours and compared them to serum Ki-67 levels in clinically healthy dogs and dogs with non-malignant diseases .Based on the promising results of this pilot study the aim of the present retrospective study was to investigate serum Ki-67 in larger groups of dogs with malignant tumours, to detect differences in regard to tumour entity, whether there is a correlation between Ki-67 and certain inflammatory cells measured in the blood and whether serum Ki-67 could be a potential pan tumour marker for future screening tests for canine malignancies.A total of 140 dogs were examined and classified into four different groups dependent on their health status: healthy, non-neoplastic diseased, benign tumours (mammary adenoma), and malignant tumours . All dogs were diagnosed between January 2018 and December 2019.n = 19) had been brought to the University of Göttingen’s veterinary clinic for small animals  for a general examination. This group of dogs had to be free of any indications of disease for at least two months prior to the clinical assessment and for four weeks thereafter. A complete blood count was performed on the respective patient and all the blood parameters had to be within the laboratory reference interval to be considered for the control group.The dogs in the healthy control group .n = 26) were brought to the veterinary clinic for small animals at the University of Göttingen  due to various illnesses. A clinical examination, diagnostic imaging , and lab work were performed on all dogs. The dogs in this group were each affected by a non-neoplastic systemic disease, such as diabetes, canine idiopathic epilepsy, chronic renal or heart failure, gastroenteritis, endometritis, urinary tract diseases, hepatitis, food poisoning, or a cervical abscess.The non-neoplastic diseased dogs , the oncology service of the veterinary clinic for small animals in Hofheim , or the veterinary clinic for gynaecology and obstetrics at the University of Gießen  due to the symptoms brought on by their particular tumour diseases. To confirm their cancer diagnoses, those dogs were subjected to all the necessary diagnostic procedures.Specialized veterinary pathologists generated histopathologic diagnoses utilizing incisional or excisional biopsy specimens of the tumours in question. Tumour volume was determined by multiplying width by height and by depth, except for lymphoma, since it is a systemic disease.n = 35; soft tissue sarcomas n = 26; malignant lymphomas n = 21).The malignant tumour group consisted of 82 dogs with different malignant tumour diseases  and analysed using a ProCyte Dx Hematology Analyzer , which counted red blood cells, white blood cells, haemoglobin and platelets, among other things. Some of these values were also used for subsequent analyses related to our measured serum Ki-67 concentrations, which are discussed in more detail below.g for 5 min. The serum was then removed from the tube and analysed using the clinical chemistry analyser  and commercial kits according to standard procedures.Standard serum tubes  were used to take serum samples for serum chemistry and then centrifuged in an Eppendorf centrifuge 5424  at 3000× g for 15 min. It was then pipetted into Eppendorf tubes, frozen at −80 °C and stored under these conditions until processed within the next 3 months.The serum for Ki-67 measurements was allowed to rest at room temperature for 2 h and then centrifuged at 1000× The Ki-67 concentrations were measured using a commercially available immunoassay kit  with non-diluted serum in duplicate, adhering to the manufacturer’s instructions.https://www.curveexpert.net/ (accessed on 17 May 2018)). According to the manufacturer, the detection range of the ELISA in serum samples is 0.625–20.0 ng/mL. The mean intra- and inter-assay coefficients are reported to be less than 15% each [First, serum samples and standards were pipetted into the appropriate wells of the microtiter plate. HRP conjugate reagent was then added and the microtiter plate was incubated at 37 °C for 1 h. The Ki-67 protein present in the samples bound to the immobilized Ki-67 antibodies on the microtiter plate, and at the same time, the detection antibody against Ki-67 contained in the HRP conjugate reagent in turn bound to the Ki-67 antigens from the samples. To remove all unbound antibodies, four manual washing steps were then performed. Subsequently, an enzymatic reaction was induced by adding chromogen solution and incubating again at 37 °C for 15 min, which became visible in the form of a blue colour complex. The resulting colour intensity is directly proportional to the amount of Ki-67 protein bound. In the final step, a stop solution was added to terminate the enzyme-substrate reaction, resulting in a colour change to yellow. The optical density (O.D.) of each well was then measured spectrophotometrically at a wavelength of 450 nm. Ki-67 concentrations in ng/mL were then calculated using the standard curve generated by the CurveExpert Professional program . Data were tested for normal distribution using the Shapiro–Wilk and the Kolmogorov–Smirnov tests, the Pearson’s correlation coefficient was used to test for linear correlation between the datasets. Non-normally distributed data were compared using the Mann–Whitney (two groups) or the Kruskal–Wallis (multiple groups) tests. Linear regression analyses were performed to examine dependency of Ki-67 on tumour volume. Receiver operation characteristic (ROC) curves were calculated for discriminating healthy dogs from tumour patients. A A wide variety of dog breeds and mixed breeds were represented and no dog breed was predominant . Informan = 19) had serum Ki-67 concentrations between 0 and 1.88 ng/mL (median of 0.29 ng/mL). The dogs in the group with non-neoplastic diseases (n = 26) suffered from metabolic diseases , neurological disorders , chronic organ failure  and inflammatory conditions . In this group Ki-67 concentrations ranged from 0.51 to 3.6 ng/mL (median 1.79 ng/mL). There was no significant difference between Ki-67 levels in dogs with metabolic disorders, neurological disorders, inflammatory conditions, or organ failure (p = 0.11), with the exception of a significantly higher Ki-67 concentration in dogs with inflammatory diseases compared to dogs with neurological disorders  and nine squamous cell carcinomas of the dermis (n = 4), oral mucosa (n = 3), and nasal mucosa (n = 2).The group of dogs with malignant tumours consisted of 82 dogs with different malignant tumour diseases that were subclassified into three groups. The carcinoma group included malignant subtypes of mammary tumours classified by the grading system established by Peña et al. 2012 (adenocarcinomas n = 26) included soft-tissue sarcomas of different anatomic locations, including subcutaneous spindle cell sarcomas (n = 17) and splenic hemangiosarcomas (n = 9). In the lymphoma group dogs with various subtypes of high-grade lymphomas were included (multicentric lymphoma (n = 18); cutaneous epitheliotropic lymphoma (n = 2), and gastrointestinal lymphoma (n = 1)).The mesenchymal tumour group .n = 9) had serum Ki-67 concentrations between 1.06 and 13.63 ng/mL (median 2.68 ng/mL). Dogs with mesenchymal tumours (n = 26) had serum Ki-67 concentrations between 0.69 and 8.27 ng/mL (median 2.35 ng/mL). Of these, subcutaneous spindle cell sarcoma (n = 17) and splenic hemangiosarcoma (n = 9) had serum Ki-67 levels between 0.69 and 8.27 ng/mL (median 2.42 ng/mL) and between 0.99 and 7.19 ng/mL (median 2.27 ng/mL), respectively. Ki-67 concentrations in dogs with high grade lymphomas (n = 21) were between 1.01 and 19.46 ng/mL (median 2.0 ng/mL). There were no statistically significant differences in Ki-67 levels between carcinoma, sarcoma, and lymphoma subgroups  had serum Ki-67 concentrations between 0.83 and 14.04 ng/mL (median 2.48 ng/mL). Dogs with squamous cell carcinomas (n = 13) serum Ki-67 concentrations ranged between 1.55 and 4.75 ng/mL (median 2.28 ng/mL).In the dogs with benign masses . No statistically significant correlation between serum Ki-67 concentrations and survival times was found .The concentrations of inflammatory cells such as neutrophils, lymphocytes, and monocytes measured in the concomitant blood test are summarized in p = 0.83, p = 0.50 and p = 0.45, respectively; p = 0.04, p = 0.03 and p = 0.02, respectively; p = 0.076, p = 0.104 and p = 0.16, respectively; Similarly, establishing ratios between serum Ki-67 concentrations and neutrophil, lymphocyte, and monocyte counts as indicators of inflammation, as well as components of the tumour microenvironment, was unable to distinguish mammary adenocarcinomas from adenomas . Using a cut-off value of 2.34 ng/mL, the sensitivity was 53.9%  and the specificity 53.9% . Using a cut-off value of 1.15 ng/mL, the sensitivity was 89.0%  and the specificity 94.7% . A cut-off value of 1.9 ng/mL resulted in a sensitivity of 63.4%  and a specificity of 61.5%  curve was calculated to discriminate dogs with benign mammary adenoma from those with malignant mammary adenocarcinoma. The area under the curve (AUC) was 0.52 , with a sensitivity of 68.8%  and a specificity of 61.5% , when using a cut-off value of 0.24  and, with a cut-off of 1.05, a sensitivity of 61.0%  and a specificity of 61.5% . Using a cut-off value of 2.15, the sensitivity and specificity was 71.4%  and 61.5% , respectively (Using ratios of serum Ki-67 concentrations and the aforementioned inflammatory parameters for the ROC curve to distinguish non-neoplastic diseased dogs from tumour-diseased dogs, the AUC improves as follows: the comparison of the Ki-67–neutrophil ratio provides an AUC of 0.73 ( of 0.24 A. Applyiectively C.Based on the WHO definition of biomarkers, a tumour marker is a measurable substance that provides information about the existence of tumour tissue in a body . In ordeKi-67 is a specific biomarker for tumour proliferation, expressed in cells during the G phase of mitosis ,33. ManyIn a pilot study investigating the expression of Ki-67 in cell cultures, to be precise cultured fibrosarcoma tumour cells, increased amounts of Ki-67 were found in culture supernatants during the proliferation phase of the cells . In anotKi-67 concentrations were also evaluated in serum, showing comparable results, although a different immunoassay kit was used than in our study . FurtherBased on these observations, we consider Ki-67 to be a valuable tumour marker indicating the presence of tumour tissue in the body. All 19 healthy patients in our study had serum Ki-67 concentrations in the lower detection range of the ELISA. Since there were no outliers in this particular group, we consider a Ki-67 serum concentration in the low picogram range to be biological for healthy dogs.The very low baseline concentrations measurable in the serum of healthy dogs is to be expected, since as a proliferation marker, a certain level of Ki-67 will also be present from rapidly proliferating normal tissues in a healthy organism, e.g., from dividing normal mucosal or bone marrow cells .In a study in human breast cancer patients, a baseline serum Ki-67 concentration was also present in the serum of the healthy control group. Similarly to our study, the level in tumour-free individuals was significantly lower than in the tumour group .The control group of diseased dogs without malignancies in this study were affected by various inflammatory, metabolic, or degenerative processes. The serum Ki-67 concentrations in this group were also significantly lower than in the group of dogs with malignancies, but were significantly higher compared to the healthy dogs. This may be explained by a proliferation of immune cells in inflammatory processes . LikewisAcute inflammatory diseases in the group of dogs suffering from non-neoplastic diseases, such as, for example, pyometra, were frequently associated with neutrophilia and monocytophilia.In case of inflammation monocyte-derived macrophages can become reinforcements for the tissue-resident macrophages ,41.Macrophages show increased proliferation in the context of inflammatory diseases and, thus, elevated Ki-67 expression, which can lead to higher serum Ki-67 concentrations . MonocytTo account for inflammation as part of the relationship between tumour tissue and the immune defence mechanisms, we calculated a ratio between the Ki-67 concentrations and the number of immune cells in peripheral blood measured in the accompanying diagnostic routine blood count. This was intended to improve the predictive value of the Ki-67 serum concentration. For example, ratios of lymphocytes to neutrophils were associated with immunohistochemically determined Ki-67 index in the following two recent human medical studies on glioma and on adrenocortical carcinoma, and elevated ratios and elevated Ki-67 indices were associated with higher pathologic grades or increased risk of tumour recurrence ,21. To tThe immunohistochemical detection of Ki-67 might be superior here . To drawIn our study, dogs with malignant tumours clearly differed from the comparison groups of healthy dogs and those with non-neoplastic diseases. This fact confirms our hypothesis that Ki-67 is elevated in patients with malignancies and, thus, can be used as a tumour marker. Furthermore, there was no correlation between serum Ki-67 concentration and tumour size. In the literature, tumour size was variably associated with Ki-67 index levels. Although two studies found no correlation with size in pituitary adenomas and soft tissue sarcomas ,45, othe9 cells for visualization [If Ki-67 elevation in malignancy is independent of tumour size, it may be useful to detect small tumours that may not yet be detectable by imaging modalities. Classical imaging modalities for tumour search reportedly require a minimum mass of 10lization . FurtherIn humans with soft tissue sarcomas, a correlation has been found between higher Ki-67 concentrations and poorer prognosis ,47. SimiLymphomas are a heterogeneous group of tumours with markedly varying but generally high Ki-67 expressions . Not surIt should be mentioned that we did not further classify our dogs with malignant lymphoma . We do nIn immunohistochemistry the Ki-67 expression is mostly evaluated within a respective tumour group, and results can vary here as well. .This restricts the utility of Ki-67 to distinguish between different tumour entities.The presence of Ki-67 only provides information about the possibility that certain cells might divide, but not about the actual proliferation rate. Therefore, additional tests are recommended, for example another marker .Our results suggest that serum Ki-67 may be useful as a non-selective screening tool for malignancy in health-checks especially in older dogs or in those with increased breed-specific cancer risk ,55. EspeBased on our data, the AUC was calculated as 0.97. Applying the Ki-67-monocyte ratio for ROC analysis to differentiate between non-neoplastic diseased dogs and dogs with malignant tumours improves the AUC up to a number of 0.74 and values above the cut-off 2.15 indicate a malignant tumour in the organism with a sensitivity of 0.71 and a specificity of 0.62.Some limitations of the present study must be addressed: We did not perform comparative measurements of Ki-67 and inflammatory parameters directly in the tumour tissue of the respective tumour patients, however this would be recommended for future follow-up studies to achieve even more informative results. As long as the direct comparative studies on Ki-67 release from the tumour tissue and the accompanying micro-tumour environment with the corresponding Ki-67 serum levels and inflammatory parameters in the blood are not yet available, we would like to label our results preliminary for the time being. Furthermore, it would be interesting to measure Ki-67 levels again after tumour therapy, and to compare these follow-up levels with the results at the time of diagnosis. To obtain even more conclusive results, follow-up studies with larger numbers of cases, more homogeneous groups, additional benign tumour types, and age-matched control animals would be recommended. This could also help to make a statement about the prognostic value of Ki-67 as a serum tumour marker.In summary, Ki-67 may be suitable as a general tumour marker to provide early indication of malignant tumour tissue present in the body during a routine examination, suggesting the need for further diagnostic testing for tumour disease, and it is recommended to also include inflammatory parameters, such as neutrophils, lymphocytes, and monocytes in these examinations.


We previously demonstrated frequent detection of HHV-6B DNA in bronchoalveolar lavage fluid (BALF) and its positive association with mortality in HCT recipients from 1992-2015 with lower respiratory tract disease (LRTD). Whether these findings remain pertinent in contemporary patients, the additive value of testing for viral gene transcription, and the correlation of HHV-6 detection in blood and BALF, are unknown.We conducted a prospective study of allogeneic HCT recipients undergoing BAL for LRTD within 120 days of HCT at three cancer centers from 2015-2019. We collected and tested paired blood and BALF for HHV-6B DNA by qPCR and HHV-6B mRNA (U38 and U90 gene transcripts) among DNA positive samples using RT-qPCR. We described the detection of HHV-6B DNA and mRNA in blood and BALF, generated receiver operating characteristic (ROC) curves to determine the ability of BALF HHV-6B DNA detection to predict HHV-6B mRNA detection, and analyzed the association of HHV-6B DNA detection with mortality.Figure 1). BALF HHV-6B DNA ≥ 218 copies/ml had an area under the curve of 0.93 for predicting detection of BALF viral mRNA . In turn, patients with BALF HHV-6B DNA ≥ 218 copies/mL had increased risk for mortality and death due to LRTD within 60 days after the BAL . This association remained after adjustment for age, oxygen use, and steroid use at the time of BAL in a multivariable Cox model .We enrolled 116 allogeneic HCT recipients who underwent 125 BALs. HHV-6B DNA was detected in 45 of 122 BALF (37%) compared to 19 of 124 (15%) plasma samples. Among the 45 BALF samples with HHV-6B DNA detected, either HHV-6B mRNA transcript was detected in 22 (49%) (HHV-6B was detected more frequently in BALF than plasma, suggesting compartment-specific reactivation. BALF HHV-6B DNA ≥ 218 copies/mL had high sensitivity and specificity for detection of viral gene transcription in BALF and was associated with increased mortality; this viral load is strikingly similar to the BALF viral load threshold of 251 copies/mL associated with mortality in our prior retrospective study. Together, these data suggest transcriptionally active HHV-6B is a clinically impactful pulmonary pathogen in contemporary HCT recipients.Joshua A. Hill, MD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Covance/CSL: Advisor/Consultant|CRISPR: Advisor/Consultant|Deverra: Grant/Research Support|Gilead: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck: Grant/Research Support|Octapharma: Advisor/Consultant|OptumHealth: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Pfizer: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant Alpana waghmare, MD, Allovir: Grant/Research Support|Ansun BioPharma: Grant/Research Support|Kyorin Pharmaceutical: Advisor/Consultant|Pfizer: Grant/Research Support|Vir/GSK: Grant/Research Support Geoffrey Hill, M.D., FRACP, FRCPA, Applied Molecular Transport: Grant/Research Support|Compass Therapeutics: Grant/Research Support|Generon Corporation: Advisor/Consultant|Heat Biologics: Grant/Research Support|iTeos Therapeutics: Advisor/Consultant|iTeos Therapeutics: Grant/Research Support|Laevoroc Oncology: Grant/Research Support|NapaJen Pharma: Advisor/Consultant|Neoleukin Therapeutics: Advisor/Consultant|Serplus Technology: Grant/Research Support|Syndax Pharmaceuticals: Grant/Research Support Danielle M. Zerr, MD MPH, AlloVir: Advisor/Consultant Sanjeet S. Dadwal, MD, FACP, FIDSA, AlloVir: Advisor/Consultant|AlloVir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|Aseptiscope: Advisor/Consultant|Aseptiscope: Stocks/Bonds|Astellas: Speaker's Bureau|Cidara: Advisor/Consultant|Gilead: Grant/Research Support|Karius: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Speaker's Bureau|Takeda: Speaker's Bureau Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Amazon: Grant/Research Support|Ansun Biopharma: Grant/Research Support|EvrysBio: Advisor/Consultant|Gates Ventures: Grant/Research Support|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Helocyte: Advisor/Consultant|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Kyorin Pharmaceuticals: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Regeneron: Grant/Research Support|ReViral: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Grant/Research Support|Vir Biotechnology: Advisor/Consultant|Vir Biotechnology: Grant/Research Support.


In this study, a theranostic approach was investigated in a TNBC xenografted mouse model by radiolabelling a close derivative of a PARPi Olaparib (termed PARPi-01) with the Auger emitters 123/125I.Triple-negative breast cancer (TNBC) lacks biomarkers for targeted therapy. Auger emitters display the best therapeutic effect, if delivered directly into the nucleus proximal to DNA. The nuclear protein Poly (ADP-ribose)-Polymerase 1 (PARP1) is a suitable target against which few inhibitors (PARPi) are clinically approved for treatment of breast cancer with germline BRCA mutation . A therapy study was performed with [125I]PARPi-01 in 4 doses . Tumour growth was monitored by CT scans longitudinally once per week. Upon reaching study endpoint, tissues were harvested and stained with TUNEL assay for detection of apoptosis induction.TNBC cell line MDA-MB-231 was subcutaneously implanted in female NOD/SCID mice. At a tumour size of ~ 500mm125I]PARPi-01 treated tumour tissues. No radiotoxicity was detected in the liver and thyroid.SPECT/CT images showed rapid hepatobiliary tracer clearance at 4 h post injection (p.i.). Retention in thyroid at 24 h p.i. suggested tracer deiodination in vivo. The tumour and liver uptake were 0.2%ID/g and 2.5%ID/g, respectively. The tumour: blood ratio was 1.3. Endogenous therapy induced a significant delay in tumour growth (doubling time increased from 8.3 to 14.2 days), but no significant survival advantage. Significantly higher apoptosis ratio was observed in [125I]PARPi-01 presents a potential radiotherapeutics for treatment of TNBC. Improvements to overcome the suboptimal pharmacokinetics are necessary for its potential clinical application.Considering the radio-cytotoxic effect in the tumour tissue and a delay on tumour doubling time, [The online version contains supplementary material available at 10.1186/s13550-022-00932-9. In BRCAmut cells, the conventional double-strand DNA damage repair mechanism homologous recombination (HR) is deficient. Although this lack of HR is a cause of tumourigenesis, it simultaneously presents an opportunity to target these cancer cells with drugs addressing other active DNA repair mechanisms. These drugs inhibit existing DNA damage repair mechanisms, which are essential for the cell survival in absence of HR, leading the cell towards apoptosis. This phenomenon of simultaneous loss of two essential repair pathways resulting in cell death is called “Synthetic lethality” MAPi  in preclinical glioblastoma and in vitro ovarian cancer models, respectively [123I]MAPi showed promising therapeutic efficacy and survival advantage upon intra-tumoural delivery in glioblastoma models. A recent study with systemic [123I]MAPi administration presented its high therapeutic efficacy in preclinical model of colorectal cancer with p53 deficiency PARPi-01 was eluted using 1 mL CH3CN and evaporated to dryness. The residue was taken up in little EtOH and diluted by isotonic saline to keep the EtOH concentration below 8%. The resulting overall radiochemical yield was 78 ± 12% with purities of > 95%. Molar activity of obtained [123I]PARPi-01 and [125I]PARPi-01 were 279 ± 216 GBq/µmol (n = 3) and 30.5 ± 6.4 GBq/µmol (n = 4), respectively.Radiolabelling was conducted as previously described  Additio: Fig. S12. Cells in culture were tested for absence of mycoplasma every two weeks.All cell lines were obtained from the Clinic for gynaecology and obstetrics, University Hospital RWTH Aachen and cultured in DMEM supplemented with 5% Fetal bovine serum and 1% Penicillin/Streptomycin. at 37 °C with 5% CO6) in a 12-well plate and treated with [125I]PARPi-01 (1 MBq/106 cells) for 24 h. Subcellular fractionation was performed using a Subcellular Fractionation kit  and Any KD Mini-Protean TGX gels (90 V for 1.5 h). The separated proteins were transferred to PVDF membranes using a wet blotting  technique. The blots were then blocked with 1% Milk/TBST and incubated with a primary antibody (anti-PARP1 (1:1000); Abcam ab227244; anti-GAPDH (1:4000); cell signalling 14C10, or anti-Histon H3 1:1000) Abcam ab1791). For the phosphorimager analysis, after blotting, the blots were sandwiched with a Fujifilm Imaging plate for Bioanalyzer (BAS-MS 2040), and incubated overnight, and the protein bound activity was imaged using a phosphorimager (Typhoon FLA 7000 version 1.2). To assess the tracer uptake, both fractions were measured using a gamma counter .Breast cancer cells were seeded (0.5 × 106 MDA-MB-231 cells were suspended in a 1:1 (volume) mixture of Culture media: Matrigel® matrix . Mice were inoculated with this cell suspension (max 200 µl) subcutaneously in the right flank. Tumour growth was monitored daily using calliper measurements. Tumour volume was calculated daily according to the formulaFemale NOD/SCID mice at 5–6 weeks of age  was used for developing subcutaneous TNBC tumour. Animals were housed in 20–24 °C in a 12 h day light cycle. For each mouse, 3 × 103, animals were proceeded for experiments. For biodistribution study animals were administered with [123I]PARPi-01 (n = 3). For therapy studies animals were randomized for control (n = 10) or [125I]PARPi-01 administration (n = 10). Randomisation was done based on equivalent distribution of tumour volumes. In addition to the daily calliper measurements image analysis of CT based tumour volumes was blinded to avoid bias. Animals were sacrificed upon completion of biodistribution study or upon reaching a humane end point for the therapy study. Humane end point is determined as reaching of either of the following conditions: 15 mm tumour diameter, 1500 mm3 tumour volume, or development of ulcers/ascites.Upon reaching of a tumour volume of 500 mm3 were intravenously administered with [123I]PARPi-01 (9.3 ± 2.7 MBq) via lateral tail-vein using a catheter under 1.5 to 2.5% isoflurane anaesthesia in oxygen at 0.8 L/min. [123I]PARPi-01 was prepared by diluting the tracer in 0.9% NaCl to a total volume of 100 μL. Administered dose was calculated by subtraction of decay-corrected syringe activity post-injection from pre-injection activity. The mice were scanned either at 4 h or at 24 h post injection (p.i.). The CT acquisition settings were as follows: default high resolution protocol, 440 uA, 50 kVp, 32 ms exposure time, and 1080° rotation in a spiral mode with 960 exposures/360°; the duration of each CT scan was 5 min. A SPECT scan was initiated at the end of each CT scan. The SPECT acquisition settings were as follows: 7-headed camera equipped with a general-purpose collimator with 28 pinholes , helical scan with 82 bed positions; the total duration of each SPECT scan was 30 min. Both CT and SPECT axial fields of view were selected at 105 mm. During the scans, the isoflurane concentration was adapted to achieve a respiratory rate between 75 and 50 breaths per minute. Images were exported and post-processed on PMOD software, version 3.8 .Imaging was performed with a small animal SPECT/CT system . Mice bearing subcutaneous MDA-MB-231 tumours upon reaching tumour volume of 500 mmFor biodistribution, animals imaged at 4 h p.i. were killed 24 h p.i., while the ones scanned 24 h p.i. were killed 5 days p.i.. Subsequently, organs were excised, weighed, and assayed for radioactivity in a gamma counter. Mean tumour and organ uptake was determined from decay-corrected tissue radioactivity normalized to injected dose and tissue sample weight (%ID/g tissue wet weight).125I]PARPi-01 (8.15 ± 2.9 MBq/dose) in four doses at 10-day intervals. The tumour volume was measured using [18F]FDG based PET/CT  once per week. [18F]FDG  was intraperitoneally administered and scanned 30 min later. Upon reaching 520 mm3 tumour volume, one additional CT imaging per week  apart from the PET/CT was performed for monitoring tumour growth.Mice were administered intravenously via lateral tail vain with Saline (0.9% NaCl) or [18F]FDG, the mice were placed on the scanner bed and the CT scan was initiated. The exposure settings used were as follows: 130 uA, 75 kVp, 230 ms exposure time, and 360° rotation with 512 views; the duration of the CT scans was ~ 5 min. A dynamic 30 min PET scan was initiated at the end of the CT scan. The CT had an axial field of view of 91.1 mm and a PET of 112 mm. During the scans, the isoflurane concentration was adapted to achieve a respiratory rate between 75–50 breaths per minute. Mice were maintained at 1% Isoflurane and heated at 37 °C from anaesthesia induction to the end of imaging. The CT images were reconstructed using a Feldkamp filtered back projection reconstruction process to a voxel size of 0.154 × 0.154 × 0.154 mm in a 592 × 592 × 560 matrix. Using vendor software, the CT values were converted into Hounsfield units (HU) using the following formulaAfter injection with PARPi-01 synthesis. SPECT/CT images obtained at 4 h p.i. show predominant tracer uptake in liver, the gastro-intestinal (GI) tract  and thyroid  followed by other organs such as stomach, colon, small intestine, and kidney. This is possibly due to the high lipophilicity of the tracer. The accumulation in tumour was 0.20 ± 0.02%ID/g  with 10 days between each dose in subcutaneous MDA-MB-231 models   and thus also the doubling time. However, Kaplan Meier-survival curve did not show any significant survival advantage upon treatment  = 0.2505)  and 15.07 days . Considering only this selection of mice, median survival time was 35 and 38 for control and therapy mice, respectively. Since, as shown for 177Lu labelled tracer, data  calculated based on SPECT images correlate with those obtained by gamma counting [Auger emitter-based therapeutic effect was assessed upon application of [ell line . 18F]FD8FFD125I]counting , liver, 125I]PARPi-01 and a non-nuclear uptake in liver and thyroid . TUNEL staining of key organs involved in the hepatobiliary excretion and in the thyroid did not show any noticeable apoptosis  [125I]PARPi-01 are necessary. The SPECT/CT image visualized a strong deiodination of [123I]PARPi-01 in vivo as indicated by the high uptake in thyroid at 24 h p.i.. The parent compound Olaparib is known to be predominantly metabolised (84%) in liver by cytochrome P450 (CYP450), which is also known to deiodinate iodine conjugated hydrocarbons. Hence, we expect this oxidative metabolism as the cause for the observed heavy deiodination of [123I]PARPi-01 [125I] in thyroid is not likely to present a therapeutic risk as it is expected to be localised in the cytoplasm after cellular uptake in the thyroid cells MAPi in healthy athymic nude mice reported the estimated absorbed dose in liver and thyroid as 0.0044 Gy/MBq and 0.0041 Gy/MBq respectively, although the intracellular distribution of [123I]MAPi was not taken into consideration KX1 that would be four times higher than the median toxic dose for Auger emitters [123I]KX1) was reported by Riad et al., to cause DNA damage in tumour tissue upon a single dose of 29.6 MBq in a subcutaneous ovarian cancer model [123I]MAPi-based study showed a therapeutic response in colorectal cancer model at 74 MBq as opposed to 296 MBq predicted by Lee et al.  in liver and thyroid and thereby a lack of radiotoxicity. Despite the observed delay in tumour growth, the difference in evaluated survival  was not significant. This is probably due to the presence of necrotic core and the longitudinal increase in the necrotic tumour volume as the tumour grows. Similarly, a lack of significance in survival was also observed in the study with [123I]MAPi in colorectal cancer model. Fractionated therapy with [123I]MAPi showed significant tumour growth delay only in p53−/− subtype, but not in a p53wt model [123I]MAPi observed in colon cancer model suggested that the DNA damaging action of Auger emitter was causing cell-death due to the synthetic lethal background provided by p53−/− status of PARP1 overexpressing tumour tissue. However, in contrast to colorectal cancer, TNBC is highly heterogenous with a wide range of genetic backgrounds. In our earlier in vitro study we reported the toxicity of [125I]PARPi-01 in a panel of representative TNBC cell lines showing mutations in different therapy-responding genes [125I]PARPi-01, however, the level of apoptosis induction remained comparable low PARPi-01 will show maximum therapeutic efficiency to predict the therapeutic effect . Another limitation of this study was the tumour exulceration, which has been also reported in a previous study [125I]PARPi-01. Nevertheless, our theranostic radiotracer ([123/125I]PARPi-01) has also shown promise considering the highly aggressive, metastatic, nature of the MDA-MB-231 cell-line and despite the low intra-tumoural tracer accumulation. The delay in tumour growth observed by our [125I]PARPi-01 therapy approach is encouraging. As visualised by Phosphor-imager with subsequent SDS/Western Blot analysis, intracellular distribution was detected in cytoplasmic as well as in the nuclear fractions . As shown for Olaparib and Talazoparib, a new formulation of the drug such as nanoencapsulation presents a promising strategy to increase its pharmacological activity [125I]PARPi-01 will allow to improve tracer´s biokinetics (reduction of liver uptake and protection from deiodination) and by this to enhance its tumoural delivery, which can be achieved by nanoparticles functionalisation with tumour addressing ligands. Even with an improved biodistribution and pharmacokinetics, not all patients will be able to benefit from this type of treatment. As previously observed, most responders to PARPi treatment were identified as DNA repair deficient, thus stratification of patients prior to therapy with PARPi is helpful for treating therapy responsive patients only [125I]PARPi-01 an initial selection of patient with HR deficiency would allow to optimally define responding patients.The radiation dose to the tumour in our model is difficult to calculate. Simulations have reported high subcellular level S values, specifically for Auger emitters such as deration . The MIRy/(Bq-s) . Goddu eiameters . Thus, eiameters . 3D-Modeemitters . Howeverer model . Also, te et al. . Due to ng genes . For thithe cell , 36. Thuus study . This mants only . Accordi125I]PARPi-01 presents a promising radiotherapeutic concept for treatment of TNBC. Despite the low intra-tumoural uptake the fractionated therapy with [125I]PARPi-01 significantly decelerated the tumour growth. The concomitant lack of off-site toxicity in normal tissues underlays the therapeutic potential of [125I]PARPi-01-based therapy. However, for clinical application further improvements regarding the tumour uptake, blood stability, and fast clearance need to be addressed. This may be done by employment of an adequate drug delivery system loaded with [125I]PARPi-01.In conclusion, [Additional file 1. Fig. S1. Reaction scheme of the radiosynthesis of [123/125I]PARPi-01. Fig S2. [18F]FDG based PET/CT therapy monitoring. Fig S3. Tumour growth curve based on CT based images for individual mice. Fig S4. TUNEL and H&E staining of tissues obtained from [125I]PARPi-01 treated mouse. Fig S5. SDS-PAGE/phosphor imaging of [125I]PARPi-01 with corresponding WB analysis of PARP1 expression


When a temperature difference is applied over a porous medium soaked with a fluid mixture, two effects may be observed, a component separation  and a pressure difference due to thermo-osmosis. In this work, we have studied both effects using non-equilibrium thermodynamics and molecular dynamics. We have derived expressions for the two characteristic parameters, the Soret coefficient and the thermo-osmotic coefficient in terms of phenomenological transport coefficients, and we show how they are related. Numerical values for these coefficients were obtained for a two-component fluid in a solid matrix where both fluid and solid are Lennard–Jones/spline particles. We found that both effects depend strongly on the porosity of the medium and weakly on the interactions between the fluid components and the matrix. The Soret coefficient depends strongly on whether the fluid is sampled from inside the porous medium or from bulk phases outside, which must be considered in experimental measurements using packed columns. If we use a methane/decane mixture in bulk as an example, our results for the Soret coefficient give that a temperature difference of 10 K will separate the mixture to about 49.5/50.5 and give no pressure difference. In a reservoir with 30% porosity, the separation will be 49.8/50.2, whereas the pressure difference will be about 15 bar. Thermo-osmotic pressures with this order or magnitude have been observed in frost-heave experiments. Thermodiffusion, the Ludwig–Soret effect, is the process by which a temperature gradient in a fluid or solid drives mass diffusion in a binary or multicomponent mixture. The effect bears the name of the scientists who discovered it, Carl Ludwig in 1865 and Charles Soret in 1879 . ResearcThermodiffusion in porous media is both challenging and interesting. Challenging, because the presence of a solid matrix adds complexity compared with bulk fluids due to the irregular pore structure and the fluid–matrix interactions , 10. IntClassic experimental techniques for measuring the Soret effect involve porous media. Packed columns were from the early 1970s a dominant experimental apparatus for measuring Soret coefficients  due to the low resistance to fluid flow, but in a porous medium it does not . A tempeThe effects of porosity were studied by Colombani et al. using a Lennard–Jones model for an equimolar methane-decane mixture with fixed “obstacles” and with interactions between the fluid and the obstacles . They foTo our knowledge, a study of a possible coupling between thermodiffusion and thermo-osmosis in a medium with two components and low porosity has not been made. The question is how the osmotic pressure affects the component separation. The effect of gravity, which gives a hydrostatic pressure gradient, was studied by Galliero and Montel , but becThe purpose of the present work is to find what effect the porous medium has on the Soret coefficient and the coupling between thermodiffusion and thermo-osmosis. For this purpose, we have used non-equilibrium molecular dynamics to simulate a porous medium soaked with a two-component fluid mixture. This is a three-component system, but the porous matrix is frozen in the sense that matrix particles do not move. The fluid is an isotope mixture, a simple and common reference case, which has been well studied by computer simulations , 8. WhenOur background and motivation for this work comes from studies of the Soret effect, which gives some emphasis on thermodiffusion. Important pioneering work on the other part of this work, thermo-osmosis, was done by Denbigh and Raumann , 29.The theory presented in Sect. k, T is the temperature, and The entropy production for coupled heat- and mass transport in a two-component fluid in a porous medium without any other external forces than a temperature gradient may be expressed as:k. The Gibbs–Duhem equation states thatk. The structure of component 3 is a FCC lattice, and the concentration of matrix particles does not vary in space. Moreover, the mole fraction i in the fluid is L-coefficients in Eqs. 15\documelux, Eq. , we get1If we express the heat flux in Eq.  with heaThe thermo-osmotic effect requires computation of the pressure, which is not well defined in a porous medium. We have therefore used a matrix configuration with bulk fluid at both sides, see Fig. mentclass2pt{minimij represent the combination of the three components, in this case the six combinations of two fluid components and one matrix component. The parameter The fluid and matrix particles were modelled with a Lennard–Jones spline potential, defined by Eq. 31\documeentclass1pt{minimaThe parameters V is the total volume of the MD box and b is the volume of a single matrix particle. The particle volume is not well defined for a soft particle like a Lennard–Jones particle, but we have used We used a tetragonal MD box with mentclass2pt{minimmentclass2pt{minimThe number of fluid particles in the matrix isThe number of fluid particles in the bulk isThe fluid densities were determined as follows. Equilibrium simulations were performed for Series A at neutral wettability, reduced temperature rom Eqs.  and 38)\documentmentclass2pt{minimSeries B was used with the same porosities and fluid densities in the porous medium as in Series A. The different overall densities is due to the contribution from the bulk in Series A, which is not relevant in Series B.x-direction into 64 layers of equal thickness. A temperature gradient was created in Series A by thermostating the fluid particles in the bulk (five layers) at each end of the MD box to a uniform high temperature The system was divided in We were also interested in the effect of wettability preferences; does a difference in wettability between fluid and matrix have an effect on the Soret coefficient? This was studied by varying the energy parameters between the fluid and the matrix, A summary of the parameter values is given in Table For each case, five parallel runs were made, each starting from a FCC structure. The matrix particles were frozen to their lattice positions. Prior to the MD runs, the fluid particle positions were randomized with different number of Monte Carlo steps. The number of MD steps was A snapshot of the system in Case 5, Series A and B, is shown in Fig. Data for temperature and composition were recorded and analyzed according to Eqs.  and 29)29) for tA typical example of mole-fraction and temperature profiles at steady state with mentclass2pt{minimS in the central part of the matrix using Eq. 29) for SThe wettability preference was controlled by the parameters S is computed from the gradients of T and Results for the Soret coefficient as function of wettability preference are given in Fig. sing Eq. , the wetThe thermo-osmotic coefficient was determined from Series A using Eq. . We founThe effect of the wettability preference is shown in Fig. Equilibrium simulations were made to clarify the effect of wettability preference in the porous medium. Figure Another aspect of the fluid distribution in the matrix is the composition. Figure The mole fraction shown in the figure is based on the two-component fluid with mentclasspt{minimaS and All equilibrium data for the mole fraction are shown in Fig. The activity coefficient of the two fluid components in the porous medium was determined from system A as follows. At equilibrium, the activity of each fluid component, Colombani et al.  found thd values –21.We found that the two configurations of the Soret cell shown in Fig. The values for the Soret coefficient shown in Figs.  studies . At 30% The main reason why System A was used, was the wish to investigate the thermo-osmotic coefficient. The definition and computation of pressure in a porous medium are topics of discussion , 34, so It is known that the thermo-osmotic coefficient can be both positive and negative . We foun∗ by Eq. , so it mst heave .S as a linear function of S (apart from factors of mole fractions and temperature) and vice versa. This shows how S and S and S as function of We found that the thermo-osmotic coefficient depends weakly on the difference in interaction strength between fluid and matrix particles , while the other filled the entire system volume (System B). Both systems were closed in the sense that the number of particles was constant during the simulations.We found that both the Soret and thermo-osmotic coefficients depend strongly on the porosity and to some extent on the two components’ ability to wet the matrix particles. The Soret coefficient, given by the ratio between the composition profile and the temperature profile, decreases monotonically from the bulk value at 100% porosity down to about 25% of the bulk value down to 30% porosity. The values at porosities above 80% are in good agreement with previous results for bulk liquids. Below 80%, systems A and B gave quite different values for the Soret coefficient, depending on where fluid samples were taken for compositional analysis. If the compositions were based on samples from the reservoirs in System A, the values were biased by the capacity of the reservoirs to sustain absorption in the matrix. With System B, the Soret coefficient was determined from the mole-fraction and temperature gradients in the matrix. We also found that the preferential absorption of the two components had a dramatic effect on the Soret coefficient in Series A, but not in Series B.The thermo-osmotic coefficient, given as the ratio between the pressure difference and the temperature difference in the two reservoirs in System B, is 0 at 100% porosity and increases non-monotonically with decreasing porosity with a dip around 40% porosity. The fluid density and mole fraction in the pores show an anomaly in the same porosity region. We have not been able to find the physical reason for this dip other than it may be related to the structure of the matrix. This illustrates that the absorption and perhaps also the permeability of the porous medium is a complex function of porosity due to the geometry of the matrix structure versus the size of the particles. In the present case, the effect of the Soret equilibrium on the thermo-osmotic coefficient is small and vice versa. The thermo-osmotic coefficients are surprisingly large, and of the same order of magnitude as has been observed in frost heave.We show that a thermodiffusion coefficient may be defined for porous systems by analogy to the definition for bulk fluids. If the porous medium is non-selective with respect to the fluid components, this definition gives the Soret coefficient by direct analogy to bulk fluids.Our findings have two important implications for experimental designs made to measure the Soret coefficient in porous media. (1) When the Soret coefficient is measured with the packed column method and fluid samples are drawn from outside the porous medium, the values may be distorted by absorption in the medium. (2) The Soret effect may depend on variations in the wettability.